Method for suppressing pathogenic mutations using programmable base editor system

Abstract

Provided herein are methods of suppressing pathogenic mutations using a programmable base editor system, and specifically, compositions comprising a polynucleotide programmable nucleotide binding domain and a nucleoside base editing domain along with a base editor of a guide polynucleotide, and methods of using the base editor. Also provided herein are base editor systems for editing nucleoside bases of a target nucleotide sequence.

Description

[0001] This invention is a divisional application of a PCT patent application entered into China with Chinese patent application number 201980046522.6, invention name “Method for curbing pathogenic mutations using a programmable base editor system” and international application date May 11, 2019.

[0002] Related Applications

[0003] This application claims the rights and interests of U.S. Provisional Application US Serial No. 62 / 670,498, filed on May 11, 2018, and US Serial No. 62 / 780,864, filed on December 17, 2018, the disclosures of which are incorporated herein by reference in their entirety. Background Art

[0004] For most well-known genetic diseases, it is necessary to correct point mutations at the target locus rather than randomly destroy genes to study or solve the potential cause of the disease. The current genome editing technology using the clustered regularly interspaced short palindromic repeats (CRISPR) system introduces double-stranded DNA breaks at the target locus as the first step of gene correction. In response to double-stranded DNA breaks, the cellular DNA repair process is mostly through non-homologous end joining, resulting in random insertions or deletions (indels) at the site of DNA division. Although most genetic diseases originate from point mutations, current correction methods for point mutations are ineffective, typically inducing a rich random insertion or deletion (indel) at the target site due to cellular response to dsDNA breaks. Therefore, there is a need for improved genome editing, which is more efficient and has far fewer undesirable products, such as random insertions or deletions (indels) or translocations.

[0005] Alpha-1 antitrypsin deficiency (A1AD) is a genetic disease in which pathogenic mutations in the SERPINA1 gene, which encodes the alpha-1 antitrypsin (A1AT) protein, result in reduced protein production in individuals with the disease. A1AT is an excellent inhibitor of neutrophil elastase, and protects tissues and organs such as the lungs from elastase degradation. As a result, elastin in the lungs of patients with A1AD is more susceptible to degradation by neutrophil elastase, and over time, loss of lung elasticity develops into chronic obstructive pulmonary disease (COPD). In healthy individuals, A1AT is produced by hepatocytes in the liver and secreted into the systemic circulation, where the protein acts as a protease inhibitor.

[0006] The most common pathogenic A1AT variant is a guanine to adenine (G→A) mutation in the SERPINA1 gene, which results in a glutamine to lysine substitution at amino acid position 342 of the A1AT protein. This substitution causes misfolding of the protein and aggregation inside hepatocytes, and ultimately, toxic aggregates lead to liver damage and cirrhosis. Although hepatotoxicity may be addressed by gene knockout (CRISPR / ZFN / TALEN) or gene knockdown (siRNA), these approaches fail to address the lung pathology. Although lung pathology can be addressed by protein supplementation therapy, this therapy does not address the liver toxicity. Gene therapy is also insufficient to address the A1AT genetic defect. Because the liver of patients with A1AD is already under a severe disease burden due to endogenous A1AT accumulation, gene therapy to increase liver A1AT will be counterproductive. Therefore, there is a need for treatments for patients with A1AD that address both the lung pathology and the liver toxicity that accompany the disease.

[0007] Incorporated by Reference

[0008] All publications, patents, and patent applications mentioned in this specification are incorporated by reference in their entirety into the disclosure of this specification, as if each publication, patent, and patent application were specifically and individually indicated as being incorporated into the disclosure of this specification. Unless otherwise indicated, all publications, patents, and patent applications mentioned in this specification are incorporated by reference in their entirety into the disclosure of this specification. Summary of the invention

[0009] SUMMARY OF THE INVENTION

[0010] Provided herein is a method for treating a genetic disorder in an individual, wherein the method comprises administering a base editor, or a polynucleotide encoding the base editor, to an individual in need thereof, wherein the base editor comprises a polynucleotide-programmable DNA binding domain and a cytidine deaminase domain; administering a guide polynucleotide to the individual, wherein the guide polynucleotide targets the base editor to a target nucleotide sequence in the individual; and when the base editor is targeted to the target nucleotide sequence, the nucleobase is edited by deaminating the nucleobase of the target nucleotide sequence, thereby treating the genetic disorder by changing the nucleobase to another nucleobase; wherein the nucleobase is in a protein coding region of the polynucleotide; and wherein the nucleobase is not the cause of the genetic disorder (i.e., the nucleobase does not code for a mutation that causes the genetic disease).

[0011] The present disclosure also provides a method for making a cell, tissue, or organ for treating a genetic disorder in an individual, wherein the method comprises contacting the cell, tissue, or organ with a base editor, or a polynucleotide encoding the base editor, wherein the base editor comprises a polynucleotide programmable DNA binding domain and a deaminase domain; contacting the cell, tissue, or organ with a guide polynucleotide, wherein the guide polynucleotide targets the base editor to a target nucleotide sequence of the cell, tissue, or organ; and when the base editor targets the target nucleotide sequence, the nucleobase of the target nucleotide sequence is edited by deaminating the nucleobase, thereby making the cell, tissue, or organ for treating the genetic disorder by changing the nucleobase to another nucleobase; wherein the nucleobase is in a protein coding region of the polynucleotide; and wherein the nucleobase is not the cause of the genetic disorder. In some embodiments, the method further comprises administering the cell, tissue, or organ to the individual. In some embodiments, the cell, tissue, or organ is autologous to the individual. In some embodiments, the cell, tissue, or organ is allogeneic to the subject. In some embodiments, the cell, tissue, or organ is xenogeneic to the subject.

[0012] In some embodiments, the result of the change of the nucleobase to another nucleobase results in an increase in the activity of the protein encoded by the polynucleotide. In some embodiments, the result of the change of the nucleobase to another nucleobase results in improved folding and / or increased stability of the protein encoded by the polynucleotide. In some embodiments, the result of the change of the nucleobase to another nucleobase results in increased expression of the protein encoded by the polynucleotide. In some embodiments, the increased expression of the protein is due to an improved translation rate of the protein. In some embodiments, the increased expression of the protein is due to an increased release rate from an organelle or cell compartment containing the protein. In some embodiments, the increased expression of the protein is due to an improved processing rate of the signal peptide of the protein. In some embodiments, the increased expression of the protein is due to an altered interaction of the protein with another protein.

[0013] In some embodiments, the nucleobase is located in a gene that is causative of the genetic disorder. In some embodiments, the editing comprises editing a plurality of nucleobases located in the gene, wherein the plurality of nucleobases are not causative of the genetic disorder. In some embodiments, the editing further comprises editing one or more additional nucleobases located in at least another gene. In some embodiments, the gene and the at least another gene encode one or more subunits of the protein. In some embodiments, the nucleobase is in a gene listed in Table 3A or Table 3B, and wherein the editing results in an amino acid change in the protein encoded by the gene indicated in Table 3A or Table 3B.

[0014] In some embodiments, the genetic disorder is retinitis pigmentosa, Usher syndrome, sickle cell disease, beta-thalassemia, alpha-1 antitrypsin deficiency (A1AD), hepatic pyrrolporphyrinosis, medium chain acyl-CoA dehydrogenase (MCAD) deficiency, lysosomal acid lipase (LAL) deficiency, phenylketonuria, hemochromatosis, von Gierke syndrome, Pompe syndrome, Gaucher syndrome, Hurler syndrome, cystic fibrosis, or chronic pain. In some embodiments, the genetic disorder is alpha-1 antitrypsin deficiency (A1AD). In some embodiments, the editing results in an amino acid change in the alpha-1 antitrypsin (A1AT) protein selected from the group consisting of F51L, M374I, A348V, A347V, K387R, T59A, and T68A. In some embodiments, the editing results in an M374I amino acid change in the A1AT protein.

[0015] In some embodiments, the genetic disorder is sickle cell disease. In some embodiments, the editing results in an amino acid change that reduces the polymerization potential of the HbA / HbS tetramer. In some embodiments, the nucleobase is located in the HBB gene encoding the beta subunit of hemoglobin (HbB). In some embodiments, the HBB gene is a sickle hemoglobin allele (HbS). In some embodiments, the editing results in an amino acid change in the beta subunit of hemoglobin. In some embodiments, the amino acid change in the beta subunit of hemoglobin comprises A70T, A70V, L88P, F85L, F85P, E22G, G16D, G16N, or any combination thereof. In some embodiments, the nucleobase is located in the HBA1 or HBA2 gene encoding the alpha subunit of hemoglobin (HbA). In some embodiments, the editing results in an amino acid change in the alpha subunit of hemoglobin. In some embodiments, the amino acid change in the α subunit is located at the polymerization interface between the α subunit and the β subunit of sickle hemoglobin. In some embodiments, the amino acid change in the α subunit of hemoglobin comprises K11E, D47G, Q54R, N68D, E116K, H20Y, H50Y, or any combination thereof.

[0016] In one aspect, compositions and methods for suppressing pathogenic mutations using programmable nucleoside base editors are provided. The present invention provides a method for treating A1AD using a base editor (e.g., BE4) to induce changes in the endogenous SERPINA1 gene. The altered SERPINA1 gene encodes the M374I mutation, which stabilizes E342K in the alpha-1 antitrypsin protein. The use of BE4 to introduce M374I can simultaneously improve liver toxicity and increase A1AT circulation to the lungs, thereby compensating for the presence of the harmful M374I mutation. This strategy simultaneously reduces the pathogenic protein load of the liver and restores functional protein to the lungs.

[0017] In another aspect, the present invention provides a method for editing a SERPINA1 polynucleotide comprising a single nucleotide polymorphism (SNP) associated with alpha-1 antitrypsin deficiency (A1AD), the method involving contacting the SERPINA1 polynucleotide with a base editor complexed with one or more guide polynucleotides, wherein the base editor comprises a polynucleotide programmable DNA binding domain and a cytidine deaminase domain, and wherein the one or more guide polynucleotides target the base editor to achieve a change in the single nucleotide polymorphism (SNP) associated with A1AD. In one embodiment, the contact is in a cell, a eukaryotic cell, a mammalian cell, or a human cell. In another embodiment, the cell is in vivo or in vitro.

[0018] In another aspect, the present invention provides a cell or a progenitor cell thereof produced by introducing a base editor, or a polynucleotide encoding the base editor, into a cell, wherein the base editor comprises a polynucleotide programmable DNA binding domain and a cytidine deaminase domain; and one or more guide polynucleotides targeting the base editor to deaminize the cytidine at nucleic acid position 1455 of the SERPINA1 polynucleotide. In one embodiment, the cell produced is a hepatocyte. In another embodiment, the cell or its progenitor cell is an embryonic cell, an induced pluripotent stem cell, or a hepatocyte. In another embodiment, the hepatocyte expresses an A1AT polypeptide. In another embodiment, the cell is from an individual suffering from A1AD. In another embodiment, the cell is a mammalian cell or a human cell.

[0019] In another aspect, the invention provides a method of treating A1AD in a subject, comprising administering to the subject a cell of any of the previous aspects. In one embodiment, the cell is autologous to the subject. In another embodiment, the cell is allogeneic to the subject.

[0020] In another aspect, the invention provides an isolated cell or population of cells proliferated or expanded from a cell of any of the previous aspects.

[0021] In another aspect, the present invention provides a method of treating A1AD in a subject, wherein the method comprises administering to the subject:

[0022] A base editor, or a polynucleotide encoding the base editor, wherein the base editor contains a polynucleotide programmable DNA binding domain and a cytidine deaminase domain; and

[0023] One or more guide polynucleotides of the base editor are targeted to effectuate a change in the cytidine at nucleic acid position 1455 of the SERPINA1 polynucleotide.

[0024] In one embodiment of the above-described aspects, the subject is a mammal or a human. In another embodiment, the method involves delivering the base editor, or a polynucleotide encoding the base editor, and the one or more guide polynucleotides to a cell of the subject. In another embodiment, the cell is a hepatocyte. In another embodiment, the cell is a hepatocyte progenitor cell. In another embodiment, the hepatocyte expresses the A1AT protein.

[0025] In another aspect, a method for producing hepatocytes or progenitor cells thereof comprises:

[0026] introducing a base editor, or a polynucleotide encoding the base editor, into a hepatocyte progenitor cell containing a SNP associated with A1AD, wherein the base editor comprises a polynucleotide programmable DNA binding domain and a cytidine deaminase domain; and one or more guide polynucleotides, wherein the one or more guide polynucleotides target the base editor to deaminize cytidine at nucleic acid position 1455 of the SERPINA1 polynucleotide, and

[0027] (b) differentiating the hepatocyte progenitor cells into hepatocytes. In one embodiment, the method involves differentiating hepatocyte progenitor cells into hepatocytes. In another embodiment, the hepatocyte progenitor cells express an A1AT polypeptide. In another embodiment, the hepatocyte progenitor cells are obtained from an individual suffering from A1AD. In another embodiment, the hepatocyte progenitor cells are mammalian cells or human cells.

[0028] In another aspect, the present invention provides a guide RNA comprising a nucleic acid sequence selected from the following:

[0029] 5'-CAAUCAUUAAGAAGACAAAGGGUUU-3'

[0030] 5'-UCAAUCAUUAAGAAGACAAAGGGUUU-3'

[0031] 5'-UUCAAUCAUUAAGAAGACAAAGGGUUU-3'

[0032] 5'-GUUCAAUCAUUAAGAAGACAAAGGGUUU-3'

[0033] 5'-UGUUCAAUCAUUAAGAAGACAAAGGGUUU-3'

[0034] 5'-UUGUUCAAUCAUUAAGAAGACAAAGGGUU-3'

[0035] 5′-UUCAAUCAUUAAGAAGACAAAG-3′

[0036] 5'-UUCAAUCAUUAAGAAGACAAAGG-3'

[0037] 5'-UCAAUCAUUAAGAAGACAAAGGG-3'

[0038] 5'-AAUCAUUAAGAAGACAAAGGGU-3'.

[0039] In another aspect, the invention provides a guide RNA comprising 18, 19, 20, 21, or 22 nucleotides of a guide RNA of an aspect set forth or otherwise described in the disclosure.

[0040] In another aspect, the present invention provides a protein-nucleic acid complex comprising a base editor of the aspects described in the present disclosure and a guide RNA as described herein.

[0041] In any of the above aspects or any other aspects of the invention as set forth in the present disclosure, the base editor deaminates the SERPINA1 polynucleotide cytidine at position 1455, thereby inducing a methionine to isoleucine mutation at amino acid position 374 of the A1AT protein. In any of the above aspects or any other aspects of the invention as set forth in the present disclosure, the A1AT polypeptide contains a lysine at amino acid position 342 and / or contains a lysine at amino acid position 376. In any of the above aspects or any other aspects of the invention as set forth in the present disclosure, the polynucleotide programmable DNA binding domain is Streptococcus pyogenes Cas9 (SpCas9) or a variant thereof. In any of the above aspects or any other aspects of the invention as set forth in the present disclosure, the SpCas9 has specificity for a PAM sequence selected from 5'-NGG-3' or 5'-GGG-3'.

[0042] In any of the above aspects or any other aspects of the invention as described in the present disclosure, the polynucleotide programmable DNA binding domain is a nuclease inactive or nickase variant. In any of the above aspects or any other aspects of the invention as described in the present disclosure, the nickase variant comprises an amino acid substitution D10A or its corresponding amino acid substitution. In any of the above aspects or any other aspects of the invention as described in the present disclosure, the cytidine deaminase domain can deaminate cytidine in deoxyribonucleic acid (DNA). In any of the above aspects or any other aspects of the invention as described in the present disclosure, the cytidine deaminase domain is a modified cytidine deaminase domain that does not occur in nature. In any of the above aspects or any other aspects of the invention as described in the present disclosure, the cytidine deaminase domain is an APOBEC deaminase domain. In any of the above aspects or any other aspects of the invention as described in the present disclosure, the base editor is BE4. In any of the above or any other aspects of the invention as described herein, the one or more guide polynucleotides contain CRISPR RNA (crRNA) and trans-micromolecule RNA (tracrRNA), wherein the crRNA comprises a nucleic acid sequence complementary to a SERPINA1 nucleic acid sequence, and the SERPINA1 nucleic acid sequence comprises a SNP associated with A1AD. In any of the above or any other aspects of the invention as described herein, the base editor is complexed with a single guide RNA (sgRNA), and the sgRNA comprises a nucleic acid sequence complementary to a SERPINA1 nucleic acid sequence encoding methionine 374.

[0043] In some embodiments, any of the methods provided herein further comprises a second editing of an additional nucleobase. In some cases, the additional nucleobase is not the cause of the genetic disorder. In some cases, the additional nucleobase is the cause of the genetic disorder.

[0044] In some embodiments, the deaminase domain is a cytidine deaminase domain or an adenosine deaminase domain. In some embodiments, the deaminase domain is a cytidine deaminase domain. In some embodiments, the deaminase domain is an adenosine deaminase domain. In some embodiments, the adenosine deaminase domain can deaminate adenine in deoxyribonucleic acid (DNA). In some embodiments, the guide polynucleotide comprises ribonucleic acid (RNA) or deoxyribonucleic acid (DNA). In some embodiments, the guide polynucleotide comprises a CRISPR RNA (crRNA) sequence, a trans-activating RNA (tracrRNA) sequence, or a combination thereof.

[0045] In some embodiments, any of the methods provided herein further comprises a second guide polynucleotide. In some embodiments, the second guide polynucleotide comprises ribonucleic acid (RNA) or deoxyribonucleic acid (DNA). In some embodiments, the second guide polynucleotide comprises a CRISPR RNA (crRNA) sequence, a trans-activating RNA (tracrRNA) sequence, or a combination thereof. In some embodiments, the second guide polynucleotide targets the base editor to a second target nucleotide sequence. In some embodiments, the polynucleotide programmable DNA binding domain comprises a Cas9 domain, a Cpf1 domain, a CasX domain, a CasY domain, a Cas12b / C2c1 domain, or a Cas12c / C2c3 domain. In some embodiments, the polynucleotide programmable DNA binding domain is nuclease dead. In some embodiments, the polynucleotide programmable DNA binding domain is a nickase. In some embodiments, the polynucleotide programmable DNA binding domain comprises a Cas9 domain. In some embodiments, the Cas9 domain comprises a nuclease-dead Cas9 (dCas9), a Cas9 nickase (nCas9), or a nuclease-active Cas9. In some embodiments, the Cas9 domain comprises a Cas9 nickase. In some embodiments, the polynucleotide programmable DNA binding domain is an engineered or modified polynucleotide programmable DNA binding domain.

[0046] In some embodiments, any of the methods provided herein further comprises a second base editor. In some embodiments, the second base editor comprises a different deaminase domain than the base editor.

[0047] In some embodiments, the editing results in less than 20% insertion or deletion (indel) formation. In some embodiments, the editing results in less than 15% insertion or deletion formation. In some embodiments, the editing results in less than 10% insertion or deletion formation. In some embodiments, the editing results in less than 5% insertion or deletion formation. In some embodiments, the editing results in less than 4% insertion or deletion formation. In some embodiments, the editing results in less than 3% insertion or deletion formation. In some embodiments, the editing results in less than 2% insertion or deletion formation. In some embodiments, the editing results in less than 1% insertion or deletion formation. In some embodiments, the editing results in less than 0.5% insertion or deletion formation. In some embodiments, the editing results in less than 0.1% insertion or deletion formation. In some embodiments, the editing does not cause translocation. BRIEF DESCRIPTION OF THE DRAWINGS

[0048] The features of the present disclosure are described in particular detail in the appended claims. A further understanding of the features and advantages of the present disclosure will be obtained by referring to the following detailed description, which describes specific embodiments in which the principles of the present disclosure are applied, and illustrates the accompanying drawings:

[0049] Figure 1 Schematic diagram comparing healthy individuals to patients with antitrypsin deficiency (A1AD). In healthy individuals, alpha-1 antitrypsin (A1AT) protects the lungs from proteases, and the liver releases alpha-1 antitrypsin into the blood. In patients with A1AD, the normal functioning A1AT protein is lacking, leading to damage to lung tissue. In addition, abnormal A1AT accumulates in liver cells, leading to cirrhosis.

[0050] Figure 2 For illustration, it shows the typical range of serum alpha-1 antitrypsin (A1AT) levels for different genotypes (normal (MM), heterozygous carriers of alpha-1 antitrypsin deficiency (MZ, SZ), and homozygous deficiency (SS, ZZ)). The serum alpha-1 antitrypsin (AAT) concentration is expressed in μM on the left Y-axis, which is commonly found in the literature. The right Y-axis shows the approximate conversion of serum AAT concentration into mg / dL units, as commonly reported in clinical laboratories and different measurement techniques (nephelometry or radial immunodiffusion).

[0051] Figure 3 Depicts the target site sequence used to introduce the repressor mutation M374I into SERPINA1. The highlighted portion is the canonical spCas9 NGG PAM, as well as target C, for which editing would result in the desired codon change M374I. Also indicated is a non-target C, which if edited would result in the undesired codon change E376K.

[0052] Figure 4It is a bar graph showing the amount of secreted protein in the culture supernatant of HEK293T cells transiently transfected with plasmids encoding different variants of the A1AT protein. The A1AT concentration was determined by ELISA as disclosed in Borel, Florie & Mueller, Christian. (2017). Alpha-1 Antitrypsin Deficiency: Methods and Protocols. 10.1007 / 978-1-4939-7163-3, the entire contents of which are incorporated herein by reference in their entirety. The two most common variants (e.g., pathogenic mutations) of A1AT are E264V (PiS allele) and E342K (PiZ allele). The abundance of PiS and PiZ protein production is lower than that of the wild-type protein. Addition of the M374I repressor mutation ( Figure 4 The M374I mutation, which is referred to as the "compensatory mutation" in the literature, apparently increases the secreted PiS and PiZ A1AT protein levels. We therefore hypothesized that the introduction of the M374I mutation using base editors and base editing methods as described herein would increase A1AT secretion from hepatocytes and simultaneously reduce hepatotoxicity and increase A1AT circulation to the lungs. A1AT: alpha-1 antitrypsin; A1AD: alpha-1 antitrypsin deficiency; "Z mutation" is the E342K (PiZ allele) mutation; "S mutation" is the E264V (PiS allele) mutation.

[0053] Figure 5 : is a bar graph showing base editing efficiency of the M374I mutation in HEK293T. Using bpNLS is superior to the SV40 nuclear localization signal. Comparing the initial codon usage, codon optimization 2 achieved higher editing efficiency when delivered both as plasmids and as mRNA+gRNA.

[0054] Figure 6 Schematic diagram showing the evolutionary strategy starting with TadA, a DNA deoxyadenosine deaminase. The E. coli stock contains a plastid stock of mutant ecTadA (TadA*) genes fused to dCas9 and a plastid requiring targeted A·T to G·C mutations to repair antibiotic resistance genes. Mutations from surviving TadA* variants entered the human ABE base editing framework.

[0055] Figure 7A graph is presented demonstrating the predicted functional elastase activity of A1AT variants that have been base edited. The graph shows the percentage of protease activity of A1AT variants with E342K (PiZ) mutation; A1AT variants with E342K (PiZ) mutation; A1AT variants with E342K mutation and compensatory M374I mutation; A1AT variants with E342K (PiZ) mutation; A1AT variants with E264V (PiS) mutation; A1AT variants with E264V mutation and compensatory M374I mutation compared to the elastase activity of wild-type (WT) A1AT.

[0056] Figures 8A to 8C Three figures are provided showing that HEK293 cells ( Fig. 8A ) and induced pluripotent stem cells (iPSC) ( Figure 8B ) Observed base editing percentage, each of which was transfected with base editor BE4. Figure 8C Shown are the percentages of editing achieved when wild-type primary hepatocytes were transfected.

[0057] Fig. 9 Shown are the percentage of base editing achieved and A1AT secretion in BE4-edited IPSC-derived hepatocytes. DETAILED DESCRIPTION

[0058] [Detailed description of preferred embodiments]

[0059] The following description and examples illustrate the implementation of the present disclosure in detail. It should be understood that this disclosure is not limited to the specific implementation described herein and can be modified. Those skilled in the art will appreciate that there are numerous variations and modifications of the present disclosure, which are encompassed within the scope of the present invention.

[0060] It should be understood that all terms are expected to be understood by those skilled in the art. Unless otherwise defined, all technical and scientific terms used herein have the same meanings as those generally understood by those skilled in the art in the relevant industry of this disclosure.

[0061]

[00136] Several embodiments disclosed herein employ, unless otherwise indicated, conventional techniques of immunology, biochemistry, chemistry, molecular biology, microbiology, cell biology, genomics, and recombinant DNA, which are within the skill of the art. For example, refer to Sambrook and Green, Molecular Cloning: A Laboratory Manual, 4th Edition (2012); the series Current Protocols in Molecular Biology (FM Ausubel, et al. eds.); the series Methods In Enzymology (Academic Press, Inc.), PCR 2: A Practical Approach (MJ MacPherson, BD Hames and GR Taylor eds. (1995)); Harlow and Lane, eds. (1988) Antibodies, A Laboratory Manual; and Culture of Animal Cells: A Manual of Basic Technique and Specialized Applications, 6th Edition (RI Freshney, ed. (2010)).

[0062] The section labels used herein are for organizational purposes only and are not to be construed as limiting the subject matter described.

[0063] Although the various features of the present disclosure may be described in the context of a single embodiment, the features may also be provided separately or in any suitable combination. Conversely, although the present disclosure is described in the context of separate embodiments for clarity, the present disclosure may also be implemented in a single embodiment.

[0064] [definition]

[0065] The following definitions supplement the industry definitions and are specific to this case, but do not refer to any related or unrelated cases, such as any commonly owned patents or patent applications. Although any similar or equivalent methods and materials described herein can be used in the testing practices disclosed herein, preferred materials and methods are described herein. Therefore, the terms used herein are only for the purpose of describing specific embodiments and are not intended to be limiting.

[0066] Unless otherwise defined, all technical and scientific terms used herein have the same definitions as those generally understood by those skilled in the art in the art to which the present invention belongs. The following references provide general definitions of many of the terms used in the present invention to those skilled in the art: Singleton et al., Dictionary of Microbiology and Molecular Biology (2nd ed. 1994); The Cambridge Dictionary of Science and Technology (Walkered., 1988); The Glossary of Genetics, 5th Ed., R. Rieger et al. (eds.), Springer Verlag (1991); and Hale & Marham, The Harper Collins Dictionary of Biology (1991).

[0067] Unless otherwise specifically stated, in this case, the use of the singular includes the plural. It should be noted that, unless the context clearly dictates otherwise, as used in the specification, the singular forms "a", "an" and "the" include the plural forms. Furthermore, the use of the term "include" and other forms such as "include", "includes" and "included" is not restrictive.

[0068] As used in the specification and claims, the terms "comprise" (and any inclusive forms, such as "comprises" and "comprises"), "have" (and any having forms, such as "have" and "has"), "include" (and any inclusive forms, such as "includes" and "include"), or "contain" (and any containing forms, such as "contains" and "contain") are inclusive or open-ended and do not exclude additional unrecited elements or method steps. It is contemplated that any implementation discussed in this specification can be implemented with respect to any method or composition of the disclosure, and vice versa. Furthermore, the compositions of the disclosure can be utilized to implement the methods of the disclosure.

[0069] The term "about" or "approximately" means within an acceptable error range for a particular value as determined by one skilled in the art, which will depend in part on how the value is measured or determined, i.e., the limits of the measurement system. For example, according to industry practice, "about" may mean within 1 or more than 1 standard deviation. Additionally, "about" may mean up to 20%, up to 10%, up to 5%, or up to 1% of a given value. Additionally, particularly with respect to biological systems or processes, the term may mean within an order of magnitude, preferably within 5 times, and more preferably within 2 times of a value. When a particular value is described in the specification and claims, unless otherwise stated, the term "about" shall be presumed to mean within an acceptable error range for the particular value.

[0070] References in the specification to "several embodiments", "one embodiment", "an embodiment", or "other embodiments" mean that a particular feature, structure, or characteristic described in connection with the embodiment is included in at least several embodiments of the present disclosure, but not necessarily all embodiments.

[0071] As used herein, "administering" means providing one or more compositions described herein to a patient or individual. By way of example and not limitation, administration (e.g., injection) of the composition may be by intravenous (iv) injection, subcutaneous (sc) injection, intradermal (id) injection, intraperitoneal (ip) injection, or intramuscular (im) injection. One or more of these routes may be used. Parenteral administration may be by bolus injection or by slow infusion over time. Alternatively or concurrently, administration may be by oral route.

[0072] "Adenosine deaminase" refers to a deaminase that catalyzes the hydrolytic deamination of adenine (A) to inosine (I). In some embodiments, the deaminase or deaminase domain is an adenosine deaminase that catalyzes the hydrolytic deamination of adenosine or deoxyadenosine to inosine or deoxyinosine, respectively. In some embodiments, the adenosine deaminase catalyzes the hydrolytic deamination of adenosine in deoxyribonucleic acid (DNA). The adenosine deaminase provided herein (e.g., genetically modified adenosine deaminase, evolved adenosine deaminase) can be from any organism, such as bacteria. In some embodiments, the adenosine deaminase is from bacteria, such as Escherichia coli (E. coli), Staphylococcus aureus (S. aureus), Salmonella typhi (S. typhi), Shewanella putrefaciens (S. putrefaciens), Haemophilus influenzae (H. influenzae), or C. crescentus (C. crescentus). In some embodiments, the adenosine deaminase is a TadA deaminase. In some embodiments, the TadA deaminase is E. coli TadA (ecTadA) deaminase or a fragment thereof.

[0073] For example, the truncated ecTadA may lack one or more N-terminal amino acids compared to the full-length ecTadA. In some embodiments, the truncated ecTadA may lack 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 6, 17, 18, 19, or 20 N-terminal amino acid residues compared to the full-length ecTadA. In some embodiments, the truncated ecTadA may lack 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 6, 17, 18, 19, or 20 C-terminal amino acid residues compared to the full-length ecTadA. In some embodiments, the ecTadA deaminase does not include an N-terminal methionine. In some embodiments, the ecTadA deaminase is an N-terminal truncated TadA. In certain embodiments, TadA can be any of the TadAs described in PCT / US2017 / 045381, which is incorporated by reference in its entirety into the disclosure of this specification.

[0074] "Agent" refers to any small molecule chemical compound, antibody, nucleic acid molecule, or polypeptide, or a fragment thereof.

[0075] "Improvement" means to reduce, arrest, weaken, diminish, stop, or stabilize the development or progression of a disease.

[0076] "Change" means that the expression level or activity of a gene or polypeptide is changed (increased or decreased) by standard industry-known methods (such as those described herein). As used in this disclosure, changes include 10% changes in expression levels, preferably 25% changes, more preferably 40% changes, and optimally 50% or more changes in expression levels.

[0077] "Analogs" refers to molecules that are not identical but have similar functional or structural characteristics. For example, a polypeptide analog retains the biological activity of the corresponding natural polypeptide while having certain biochemical modifications that enhance the analog's function compared to the natural polypeptide. Such biochemical modifications can increase the analog's protease resistance, membrane permeability, or half-life without altering, for example, ligand binding. Analogs can include non-natural amino acids.

[0078] "Alpha-1 antitrypsin (A1AT) protein" means a polypeptide or fragment thereof having at least about 95% amino acid sequence identity to UniProt Accession No. P01009. In certain embodiments, the A1AT protein comprises one or more alterations compared to the following reference sequences. In a specific embodiment, the A1AT protein associated with A1AD comprises the E342K mutation. Examples of A1AT amino acid sequences are provided below.

[0079]

[0080] The term "base editor (BE)" refers to an agent comprising a polypeptide that can modify a nucleobase (e.g., A, T, C, G, or U) within a nucleic acid sequence (e.g., DNA or RNA). In some embodiments, the base editor is a fusion protein comprising a polynucleotide programmable nucleotide binding domain and a nucleobase editing domain (e.g., a cytidine deaminase domain or an adenosine deaminase domain) together with a guide polynucleotide (e.g., a guide RNA). In some embodiments, the base editor is a cytidine base editor (CBE). In some embodiments, the base editor is an adenosine base editor (ABE). In some embodiments, the polynucleotide programmable DNA binding domain is fused to or linked to a deaminase domain. In some embodiments, the base editor comprises a polynucleotide programmable DNA binding domain and a deaminase domain together with a guide polynucleotide (e.g., a guide RNA). In some embodiments, the polynucleotide programmable DNA binding domain is a CRISPR-associated (e.g., Cas or Cpf1) enzyme. In some embodiments, the base editor is a Cas9 protein fused to a deaminase domain (e.g., an adenosine deaminase or a cytidine deaminase). In some embodiments, the base editor is a catalytically dead Cas9 (dCas9) fused to a deaminase domain. In some embodiments, the base editor is a Cas9 nickase (nCas9) fused to a deaminase domain. In some embodiments, the base editor is fused to a base excision repair (BER) inhibitor. In some embodiments, the base excision repair inhibitor is a uracil DNA glycosylation inhibitor (UGI). In some embodiments, the base excision repair inhibitor is an inosine base excision repair inhibitor. In some embodiments, the base editor can deaminize bases inside nucleic acids. In some embodiments, the base editor can deaminize bases inside DNA molecules. In some embodiments, the base editor can deaminize bases inside RNA molecules. In some embodiments, the base editor can deaminize adenine (A). In some embodiments, adenosine deaminase is evolved from TadA. In some embodiments, the base editor can deaminate guanine (G). In some embodiments, the base editor can deaminate adenine (A). In some embodiments, the base editor can deaminate cytosine (C). The details of the base editor are described in International PCT Application Nos. PCT / 2017 / 045381 (WO2018 / 027078) and PCT / US2016 / 058344 (WO2017 / 070632), each of which is incorporated by reference into the disclosure of this specification.See also Komor, AC, et al., “Programmable editing of a target base in genomic DNA without double-stranded DNA cleavage” Nature 533, 420-424 (2016); Gaudelli, NM, et al., “Programmable base editing of A·T to G·C in genomic DNA without DNA cleavage” Nature 551, 464-471 (2017); and Komor, AC, et al., “Improved base excision repair inhibition and bacteriophage Mu Gam protein yields C:G-to-T:A base editor with higher efficiency and product purity” editors with higher efficiency and product purity”) Science Advances 3:eaao4774 (2017), the full text of which is incorporated into the disclosure of this specification by reference.

[0081] In some embodiments, the cytidine base editor BE4 used in the base editing compositions, systems and methods described herein has the following nucleic acid sequence (8877 base pairs), (Addgene, Watertown, MA.; Komor AC, et al., 2017, Sci Adv., 30; 3(8):eaao4774.doi:10.1126 / sciadv.aao4774) provided below. Polynucleotide sequences having at least 95% or more identity to the BE4 nucleic acid sequence are also contemplated.

[0082]

[0083]

[0084]

[0085]

[0086] In some embodiments, the cytidine base editor has the following sequence:

[0087]

[0088]

[0089]

[0090] In some embodiments, the cytidine base editor has the following sequence:

[0091]

[0092]

[0093]

[0094]

[0095] "Base editing activity" refers to the action to chemically change the base inside the nucleotide. In one embodiment, the first base is converted to a second base. In one embodiment, the base editing activity is cytidine deaminase activity, for example, converting the target C·G to T·A. In another embodiment, the base editing activity is adenosine deaminase activity, for example, converting the target A·T to G·C.

[0096] The term "base editor system" refers to a system for editing nucleobases of a target nucleotide sequence. In some embodiments, the base editor system comprises (1) a base editor (BE) comprising a polynucleotide programmable nucleotide binding domain and a deaminase domain for deaminating a nucleobase; and (2) a guide polynucleotide (e.g., a guide RNA) together with a polynucleotide programmable nucleotide binding domain. In some embodiments, the polynucleotide programmable nucleotide binding domain is a polynucleotide programmable DNA binding domain. In some embodiments, the base editor is a cytidine base editor (CBE). In some embodiments, the base editor is an adenosine base editor (ABE).

[0097] In some embodiments, a nucleoside base editor system may include more than one base editing component. For example, a nucleoside base editor system may include more than one deaminase. In some embodiments, a nuclease base editor system may include one or more cytidine deaminases and / or one or more adenosine deaminases. In some embodiments, a single guide polynucleotide may be used to target different deaminases to a target nucleic acid sequence. In some embodiments, a single pair of guide polynucleotides may be used to target different deaminases to a target nucleic acid sequence.

[0098] The nucleobase components and polynucleotide programmable nucleotide binding components of the base editor system can be covalently or non-covalently or covalently associated with each other. For example, in some embodiments, the deaminase domain can be targeted to the target nucleotide sequence through the polynucleotide programmable nucleotide binding domain. In some embodiments, the polynucleotide programmable nucleotide binding domain can be fused or linked to the deaminase domain. In some embodiments, the polynucleotide programmable nucleotide binding domain can target the deaminase domain to the target nucleotide sequence by non-covalently interacting or associating with the deaminase domain. For example, in some embodiments, the nucleobase editing component, such as the deaminase component, may include an additional heterologous portion or domain, which can interact, associate, or form a complex with an additional heterologous portion or domain that is part of the polynucleotide programmable nucleotide binding domain. In some embodiments, the additional heterologous portion can bind to, interact, associate, or form a complex with a polypeptide. In some embodiments, the additional heterologous portion can bind to, interact, associate, or form a complex with a polynucleotide. In some embodiments, the additional heterologous moiety can be bound to a guide polynucleotide. In some embodiments, the additional heterologous moiety can be bound to a polypeptide linker. In some embodiments, the additional heterologous moiety can be bound to a polynucleotide linker. The additional heterologous moiety can be a protein domain. In some embodiments, the additional heterologous moiety can be a K homology (KH) domain, an MS2 coat protein domain, a PP7 coat protein domain, a SfMu Com coat protein domain, a sterile alpha motif, a telomerase Ku binding motif and Ku protein, a telomerase Sm7 binding motif and Sm7 protein, or an RNA recognition motif.

[0099] The base editor system may further include a guide polynucleotide component. It should be understood that the components of the base editor system can be associated with each other by covalent bonds, non-covalent interactions, or any combination of their association and interaction. In some embodiments, the deaminase domain can be targeted to the target nucleotide sequence by a guide polynucleotide. For example, in some embodiments, the nucleoside base editing component of the base editor system, such as a deaminase component, may include an additional heterologous portion or domain (e.g., a polynucleotide binding domain, such as an RNA or DNA binding protein), which can interact, associate, or form a complex with a portion or segment of the guide polynucleotide (e.g., a polynucleotide motif). In some embodiments, an additional heterologous portion or domain (e.g., a polynucleotide binding domain, such as an RNA or DNA binding protein) can be fused or linked to the deaminase domain. In some embodiments, the additional heterologous portion can bind to, interact with, associate with, or form a complex with a polypeptide. In some embodiments, the additional heterologous portion can bind to, interact with, associate with, or form a complex with a polynucleotide. In some embodiments, the additional heterologous moiety can be bound to a guide polynucleotide. In some embodiments, the additional heterologous moiety can be bound to a polypeptide linker. In some embodiments, the additional heterologous moiety can be bound to a polynucleotide linker. The additional heterologous moiety can be a protein domain. In some embodiments, the additional heterologous moiety can be a K homology (KH) domain, an MS2 coat protein domain, a PP7 coat protein domain, a SfMu Com coat protein domain, a sterile alpha motif, a telomerase Ku binding motif and Ku protein, a telomerase Sm7 binding motif and Sm7 protein, or an RNA recognition motif.

[0100] In some embodiments, the base editor system may further include a base excision repair (BER) inhibitor component. It should be understood that the components of the base editor system can be associated with each other by covalent bonds, non-covalent interactions, or any combination of their association and interaction. The BER inhibitor component may include a base excision repair inhibitor. In some embodiments, the base excision repair inhibitor can be a uracil DNA glycosylation inhibitor (UGI). In some embodiments, the base excision repair inhibitor can be an inosine base excision repair inhibitor. In some embodiments, the base excision repair inhibitor can be targeted to a target nucleotide sequence through a polynucleotide programmable nucleotide binding domain. In some embodiments, the polynucleotide programmable nucleotide binding domain can be fused or linked to a base excision repair inhibitor. In some embodiments, the polynucleotide programmable nucleotide binding domain can be fused or linked to a deaminase domain and a base excision repair inhibitor. In some embodiments, the polynucleotide programmable nucleotide binding domain can target the base excision repair inhibitor to a target nucleotide sequence by non-covalently interacting or associating with the base excision repair inhibitor. For example, in some embodiments, the base excision repair inhibitor may include an additional heterologous portion or domain that can interact, associate, or form a complex with an additional heterologous portion or domain that is part of a programmable nucleotide binding domain of a polynucleotide. In some embodiments, the base excision repair inhibitor can be targeted to a target nucleotide sequence by a guide polynucleotide. For example, in some embodiments, the base excision repair inhibitor can include an additional heterologous portion or domain (e.g., a polynucleotide binding domain, such as an RNA or DNA binding protein) that can interact, associate, or form a complex with a portion or segment of a guide polynucleotide (e.g., a polynucleotide motif). In some embodiments, the additional heterologous portion or domain of the guide polynucleotide (e.g., a polynucleotide binding domain, such as an RNA or DNA binding protein) can be fused or linked to the base excision repair inhibitor. In some embodiments, the additional heterologous portion can bind to, interact with, associate with, or form a complex with the polynucleotide. In some embodiments, the additional heterologous portion can bind to, bind to, interact with, associate with, or form a complex with the guide polynucleotide. In some embodiments, the additional heterologous moiety can be bound to a guide polynucleotide. In some embodiments, the additional heterologous moiety can be bound to a polypeptide linker. In some embodiments, the additional heterologous moiety can be bound to a polynucleotide linker. The additional heterologous moiety can be a protein domain. In some embodiments, the additional heterologous moiety can be a K homology (KH) domain, an MS2 coat protein domain, a PP7 coat protein domain, a SfMu Com coat protein domain, a sterile alpha motif, a telomerase Ku binding motif and Ku protein, a telomerase Sm7 binding motif and Sm7 protein, or an RNA recognition motif.

[0101] The term "Cas9" or "Cas9 domain" refers to an RNA-guided nuclease that includes a Cas9 protein or a fragment thereof (e.g., a protein that includes an active, inactive, or partially active DNA cleavage domain of Cas9, and / or a gRNA binding domain of Cas9). Cas9 nucleases are occasionally referred to as casn1 nucleases or CRISPR (clustered regularly interspaced short palindromic repeats) associated nucleases. An example of Cas9 is Streptococcus pyogenes Cas9, whose amino acid sequence is provided below

[0102]

[0103] (Single lower line: HNH domain; double lower line: RuvC domain).

[0104] The term "conservative amino acid substitution" or "conservative mutation" refers to the replacement of one amino acid by another amino acid with common properties. A functional way to define common properties between individual amino acids is to analyze the normalized frequencies of amino acid changes between corresponding proteins of homologous organisms (Schulz, GE and Schirmer, RH, Principles of Protein Structure, Springer-Verlag, New York (1979)). Based on these analyses, groups of amino acids can be defined in which the amino acids within a group are preferentially exchanged with each other and thus have the most similar effects on the overall protein structure (Schulz, GE and Schirmer, RH, supra). Non-limiting examples of conservative mutations include amino acid substitutions of amino acids, for example, lysine for arginine, and vice versa, so that a positive charge is maintained; glutamic acid for aspartic acid, and vice versa, so that a negative charge is maintained; serine for threonine, so that a free -OH is maintained; and glutamic acid for asparagine, so that a free -NH is maintained. 2 .

[0105] The term "Cas9" or "Cas9 domain" refers to an RNA-guided nuclease that comprises a Cas9 protein or a fragment thereof (e.g., a protein that comprises an active, inactive, or partially active DNA cleavage domain of Cas9, and / or a gRNA binding domain of Cas9). Cas9 nucleases are occasionally referred to as casn1 nucleases or CRISPR (clustered regularly interspaced short palindromic repeats) associated nucleases. An example of Cas9 is Streptococcus pyogenes Cas9, whose amino acid sequence is provided as follows

[0106] (Single lower line: HNH domain; double lower line: RuvC domain).

[0107] The terms "coding sequence" or "protein coding sequence" are used interchangeably in this disclosure and refer to a polynucleotide segment that codes for a protein. The boundaries of the region or sequence are closer to the 5' end with the start codon and closer to the 3' end with the stop codon. The coding sequence is also called an open reading frame.

[0108] The term "conservative amino acid substitution" or "conservative mutation" refers to the replacement of one amino acid by another amino acid with common properties. A functional way to define common properties between individual amino acids is to analyze the normalized frequencies of amino acid changes between corresponding proteins of homologous organisms (Schulz, GE and Schirmer, RH, Principles of Protein Structure, Springer-Verlag, New York (1979)). Based on these analyses, groups of amino acids can be defined in which the amino acids within a group are preferentially exchanged with each other and thus have the most similar effects on the overall protein structure (Schulz, GE and Schirmer, RH, supra). Non-limiting examples of conservative mutations include amino acid substitutions of amino acids, for example, lysine for arginine, and vice versa, so that a positive charge is maintained; glutamic acid for aspartic acid, and vice versa, so that a negative charge is maintained; serine for threonine, so that a free -OH is maintained; and glutamic acid for asparagine, so that a free -NH is maintained. 2 .

[0109] "Cytidine deaminase" refers to a polypeptide or fragment thereof that catalyzes a deamination reaction to convert an amino group to a carbonyl group. In one embodiment, a cytidine deaminase converts cytosine to uracil, or converts 5-methylcytosine to thymine. PmCDA1, which is derived from Petromyzon marinus (PmCDA1); AID (activation-induced cytidine deaminase, AICDA), which is derived from mammals (e.g., humans, pigs, cows, horses, monkeys, etc.); and APOBEC are examples of cytidine deaminase.

[0110] As used in this disclosure, the term "deaminase" or "deaminase domain" refers to a protein or enzyme that catalyzes a deamination reaction. In some embodiments, the deaminase or deaminase domain is a cytidine deaminase, which catalyzes the hydrolytic deamination of cytidine or deoxycytidine to uridine or deoxyuridine, respectively. In some embodiments, the deaminase or deaminase domain is a cytidine deaminase, which catalyzes the hydrolytic deamination of cytosine to uracil. In some embodiments, the deaminase is an adenosine deaminase, which catalyzes the hydrolytic deamination of adenine to hypoxanthine.

[0111] In some embodiments, the deaminase or deaminase domain is a variant of a naturally occurring deaminase from an organism such as a human, chimpanzee, orangutan, monkey, cow, dog, rat, or mouse. In some embodiments, the deaminase or deaminase domain does not occur in nature. For example, in some embodiments, the deaminase or deaminase domain is at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.1%, at least 99.2%, at least 99.3%, at least 99.4%, at least 99.5%, at least 99.6%, at least 99.7%, at least 99.8%, or at least 99.9% identical to a naturally occurring deaminase. For example, the deaminase domain is described in International PCT Application Nos. PCT / 2017 / 045381 (WO2018 / 027078) and PCT / US2016 / 058344 (WO2017 / 070632), each of which is incorporated by reference into the disclosure of this specification in its entirety. See also Komor, AC, et al., “Programmable editing of target bases in genomic DNA without double-stranded DNA cleavage,” Nature 533, 420-424 (2016); Gaudelli, NM, et al., “Programmable base editing of A·T to G·C in genomic DNA without DNA cleavage,” Nature 551, 464-471 (2017); and Komor, AC, et al., “Improved base excision repair inhibition and phage μGam protein with higher efficiency and product purity to obtain C:G-to-T:A base editors,” Science Advances 3:eaao4774 (2017); and Rees, HA, et al., “Base editing: precision chemistry on the genome and transcriptome of living cells.” Nat Rev Genet. 2018 Dec; 19(12): 770-788. doi: 10.1038 / s41576-018-0059-1, the entire contents of which are incorporated into the disclosure of this specification by reference.

[0112] "Detectable label" refers to a composition that, when linked to a molecule of interest, enables the latter to be detected by spectroscopic, photochemical, biochemical, immunochemical, or chemical means. For example, useful labels include radioisotopes, magnetic beads, metal beads, colloidal particles, fluorescent dyes, electron-dense reagents, enzymes (e.g., commonly used in ELISA), biotin, digoxigenin, or haptens.

[0113] "Disease" means any condition or disorder that damages or interferes with the normal function of a cell, tissue, or organ. Examples of diseases include retinitis pigmentosa, Usher syndrome, sickle cell disease, beta-thalassemia, alpha-1 antitrypsin deficiency (A1AD), hepatic pyrroloporphyrinosis, medium chain acyl-CoA dehydrogenase (MCAD) deficiency, lysosomal acid lipase (LAL) deficiency, phenylketonuria, hemochromatosis, von Gierke's syndrome, Pompe syndrome, Gaucher syndrome, Hurler syndrome, cystic fibrosis, or chronic pain. In certain embodiments, the disease is A1AD.

[0114] An "effective amount" refers to the amount of an agent or active compound (e.g., a base editor as described herein) required to improve disease symptoms compared to an untreated patient. The effective amount of an active compound used in the practice of the present invention to therapeutically treat a disease varies depending on the method of administration, age, weight, and general health of the individual. Ultimately, a clinician or veterinarian will determine the appropriate dosage. This amount is referred to as an "effective" amount. In one embodiment, an effective amount is an amount of a base editor of the present invention sufficient to introduce a gene change into a cell (e.g., a cell in vitro or in vivo). In one embodiment, the effective amount is the amount of base editor required to achieve a therapeutic effect (e.g., alleviating or controlling retinitis pigmentosa, Usher syndrome, sickle cell disease, β-thalassemia, alpha-1 antitrypsin deficiency (A1AD), hepatic pyrroloporphyrinosis, medium chain acyl-CoA dehydrogenase (MCAD) deficiency, lysosomal acid lipase (LAL) deficiency, phenylketonuria, hemochromatosis, von Gierke syndrome, Pompe syndrome, Gaucher syndrome, Hurler syndrome, cystic fibrosis, or chronic pain). This therapeutic effect does not need to be sufficient to change the pathogenic gene in all cells of an individual, tissue, or organ, but only changes the pathogenic gene in about 1%, 5%, 10%, 25%, 50%, 75% or more of the cells of the individual, tissue, or organ. In one embodiment, the effective amount is sufficient to ameliorate one or more symptoms of a disease (e.g., retinitis pigmentosa, Usher syndrome, sickle cell disease, β-thalassemia, alpha-1 antitrypsin deficiency (A1AD), hepatic pyrroloporphyrinosis, medium chain acyl-CoA dehydrogenase (MCAD) deficiency, lysosomal acid lipase (LAL) deficiency, phenylketonuria, hemochromatosis, von Gierke's syndrome, Pompe syndrome, Gaucher syndrome, Hurler syndrome, cystic fibrosis, or chronic pain).

[0115] A "fragment" refers to a portion of a polypeptide or nucleic acid molecule. Such a portion preferably contains at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, or 90% of the full length of a reference nucleic acid molecule or polypeptide. A fragment may contain 10, 20, 30, 40, 50, 60, 70, 80, 90, or 100, 200, 300, 400, 500, 600, 700, 800, 900, or 1000 nucleotides or amino acids.

[0116] "Hybridization" refers to hydrogen bonding between complementary nucleobases, which can be Watson-Crick, Hoostein, or anti-Hoostein hydrogen bonding. For example, adenine and thymine are complementary nucleobases that form pairs through hydrogen bonding.

[0117] The term "inhibitor of base repair", "base repair inhibitor", or grammatical synonyms thereof refers to a protein that can inhibit the activity of a nucleic acid repair enzyme (e.g., a base excision repair enzyme). Non-limiting examples of base repair inhibitors include inhibitors of APE1, Endo III, Endo IV, Endo V, Endo VIII, Fpg, hOGG1, hNEIL1, T7 Endol, T4PDG, UDG, hSMUG1, and hAAG. In some embodiments, the base repair inhibitor is an inhibitor of Endo V or hAAG. In some embodiments, the base repair inhibitor is a catalytically inactive Endo V or a catalytically inactive hAAG. In some embodiments, the base repair inhibitor is a uracil glycosylase inhibitor (UGI). UGI refers to a protein that can inhibit a uracil DNA glycosylase base excision repair enzyme. In some embodiments, the UGI domain comprises a wild-type UGI or a wild-type UGI fragment. In some embodiments, the UGI protein provided herein comprises a UGI fragment and a protein homologous to a UGI or UGI fragment. In some embodiments, the base repair inhibitor is an inosine base excision repair inhibitor. In some embodiments, the base repair inhibitor is a "catalytically inactive inosine-specific nuclease" or a "dead inosine-specific nuclease".

[0118] Without wishing to be bound by any particular theory, catalytically inactive inosine glycosylases (e.g., alkyladenine glycosylase (AAG)) can bind inosine but cannot generate an abasic site or remove inosine, thereby sterically blocking the newly generated inosine moiety from the DNA damage / repair mechanism. In some embodiments, the catalytically inactive inosine-specific nuclease can bind inosine in nucleic acids but will not cleave nucleic acids. Non-limiting examples of catalytically inactive inosine-specific nucleases include, for example, catalytically inactive alkyladenine glycosylase (AAG nuclease) from humans, and, for example, catalytically inactive endonuclease V (EndoV nuclease) from Escherichia coli. In some embodiments, the catalytically inactive AAG nuclease comprises an E125Q mutation or a corresponding mutation in another AAG nuclease.

[0119] The terms "isolated," "purified," or "biologically pure" refer to materials that are free to varying degrees from components normally associated with those found in nature. "Isolated" indicates a degree of separation from an original source or surrounding environment. "Purification" indicates a higher degree of separation than isolation. A "purified" or "biologically pure" protein is sufficiently free of other materials such that any impurities do not substantially affect the biological properties of the protein, or cause other undesirable consequences. In other words, a nucleic acid or peptide of the invention is purified if it is substantially free of cellular material, viral material, or culture medium when produced by recombinant DNA techniques, or substantially free of chemical precursors or other chemicals when chemically synthesized. Purity and homogeneity are typically determined using analytical chemistry techniques, such as polyacrylamide gel electrophoresis or high performance liquid chromatography. The term "purified" indicates that the nucleic acid or protein forms essentially a single band in an electrophoretic gel. For proteins that can undergo modifications, such as phosphorylation or glycosylation, different modifications can produce different isolated proteins that can be separately purified.

[0120] "Isolated polynucleotide" means a nucleic acid (e.g., DNA) that is free of genes that are adjacent to the native genome of the organism from which the nucleic acid molecule of the invention is derived. Thus, the term includes, for example, recombinant DNA that is incorporated into a vector; incorporated into a self-replicating plasmid or virus; or incorporated into the genomic DNA of a prokaryotic or eukaryotic organism; or that exists as a separate molecule independent of other sequences (e.g., cDNA or genomic or cDNA fragments generated by PCR or restriction endonuclease digestion). In addition, the term includes RNA molecules transcribed from a DNA molecule, as well as recombinant DNA that is part of a fusion gene encoding an additional polypeptide sequence.

[0121] "Isolated polypeptide" means a polypeptide of the present invention that has been separated from naturally associated components. Typically, a polypeptide is isolated when it is at least 60% free by weight of the proteins and naturally occurring organic molecules with which it is naturally associated. Preferably, the preparation is at least 75%, more preferably at least 90%, and most preferably at least 99% by weight of the polypeptide of the present invention. An isolated polypeptide of the present invention can be obtained, for example, by extraction from a natural source, or by expression of a recombinant nucleic acid encoding the polypeptide, or by chemically synthesizing the protein. Purity can be measured by any suitable method, for example, by column chromatography, polyacrylamide gel electrophoresis, or by HPLC analysis.

[0122] As used in the present disclosure, the term "linker" may refer to a covalent linker (e.g., a covalent bond), a non-covalent linker, a chemical group, or a molecule that links two molecules or parts, such as two components of a protein complex or a ribonucleoside complex, or two domains of a fusion protein, such as a polynucleotide programmable DNA binding domain (e.g., dCas9) and a deaminase domain (e.g., adenosine deaminase or cytidine deaminase). The linker can join different components or different parts of components of a base editor system. For example, in some embodiments, the linker can join the guide polynucleotide binding domain of the polynucleotide programmable nucleotide binding domain and the catalytic domain of the deaminase. In some embodiments, the linker can join the CRISPR polypeptide and the deaminase. In some embodiments, the linker can join Cas9 and the deaminase. In some embodiments, the linker can join dCas9 and the deaminase. In some embodiments, the linker can join nCas9 and the deaminase. In some embodiments, the linker can join the deamination component of the base editor system and the polynucleotide programmable nucleotide binding component. In some embodiments, the linker can bind the RNA binding portion of the deamination component of the base editor system to the polynucleotide programmable nucleotide binding component. In some embodiments, the linker can bind the RNA binding portion of the deamination component of the base editor system to the RNA binding portion of the polynucleotide programmable nucleotide binding component. The linker can be located between two groups, molecules, or other parts, or there are two groups, molecules, or other parts next to each other, and are linked to each other by covalent bonds or non-covalent interactions, thereby linking the two. In some embodiments, the linker can be an organic molecule, group, polymer, or chemical part. In some embodiments, the linker can be a polynucleotide. In some embodiments, the linker can be a DNA linker. In some embodiments, the linker can be an RNA linker. In some embodiments, the linker can include a nucleic acid aptamer that can bind to a ligand. In some embodiments, the linker can be a carbohydrate, a peptide, a protein, or a nucleic acid. In some embodiments, the linker can include a nucleic acid aptamer derived from a riboswitch. The riboswitch from which the aptamer is derived can be selected from: theophylline riboswitch, thiamine pyrophosphate (TPP) riboswitch, adenosylcobalamin (AdoCbl) riboswitch, S-adenosylmethionine (SAM) riboswitch, SAH riboswitch, riboflavin mononucleotide (FMN) riboswitch, tetrahydrofolate riboswitch, lysine riboswitch, glycine riboswitch, purine riboswitch, GlmS riboswitch, or pre-Q nucleoside 1 (PreQ1) riboswitch. In some embodiments, the linker can comprise a riboswitch capable of binding to a polypeptide or protein domain, such as a polypeptide linker.In some embodiments, the linker can be a K homology (KH) domain, an MS2 coat protein domain, a PP7 coat protein domain, a SfMu Com coat protein domain, a sterile alpha motif, a telomerase Ku binding motif and Ku protein, a telomerase Sm7 binding motif and Sm7 protein, or an RNA recognition motif. In some embodiments, the polypeptide linker can be part of a base editor system component. For example, a nucleoside base editing component can include a deaminase domain and an RNA recognition motif.

[0123] In some embodiments, the linker can be an amino acid or a plurality of amino acids (e.g., a peptide or protein). In some embodiments, the linker can be about 5-100 amino acids in length, for example, about 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 20-30, 30-40, 40-50, 50-60, 60-70, 70-80, 80-90, or 90-100 amino acids in length. In some embodiments, the linker can be about 100-150, 150-200, 200-250, 250-300, 300-350, 350-400, 400-450, or 450-500 amino acids in length. It is contemplated that longer or shorter linkers are also contemplated.

[0124] In some embodiments, the linker joins the gRNA binding domain of the RNA programmable nuclease (including the Cas9 nuclease domain) and the catalytic domain of the nucleic acid editing protein (e.g., cytidine deaminase or adenosine deaminase). In some embodiments, the linker joins dCas9 and the nucleic acid editing protein. For example, the linker is located between two groups, molecules, or other parts, or is flanked by two groups, molecules, or other parts, and is linked to each other by a covalent bond, thereby linking the two. In some embodiments, the linker is an amino acid or a plurality of amino acids (e.g., a peptide or protein). In some embodiments, the linker is an organic molecule, group, polymer, or chemical part. In some embodiments, the linker is 5-200 amino acids in length, e.g., 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 25, 35, 45, 50, 55, 60, 60, 65, 70, 70, 75, 80, 85, 90, 90, 95, 100, 101, 102, 103, 104, 105, 110, 120, 130, 140, 150, 160, 175, 180, 190, or 200 amino acids in length. Longer or shorter linkers are also contemplated. In some embodiments, the linker comprises the amino acid sequence SGSETPGTSESATPES, which may also be referred to as an XTEN linker. In some embodiments, the linker comprises the amino acid sequence SGGS. In some embodiments, the linker comprises (SGGS) n 、(GGGS) n 、(GGGGS) n , (G) n , (EAAAK) n 、(GGS) n , SGSETPGTSESATPES, or (XP) n motif, or any combination thereof, wherein n is independently an integer from 1 to 30, and wherein X is any amino acid. In some embodiments, n is 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15. In some embodiments, the linker comprises a plurality of proline residues and is 5-21, 5-14, 5-9, 5-7 amino acids long, for example, PAPAP, PAPAPA, PAPAPAP, PAPAPAPA, P(AP) 4 、P(AP) 7 、P(AP) 10 These proline-rich linkers are also called "rigid" linkers.

[0125] In some embodiments, the domains of the base editor are fused via a linker comprising an amino acid sequence of SGGSSGSETPGTSESATPESSGGS, SGGSSGGSSGSETPGTSESATPESSGGSSGGS, or GGSGGSPGSPAGSPTSTEEGTSESATPESGPGTSTEPSEGSAPGSPAGSPTSTEEGTSTEPSEGSAPGTSTEPSEGSAPGTSESATPESGPGSEPATSGGSGGS. In some embodiments, the domains of the base editor are fused via a linker comprising the amino acid sequence SGSETPGTSESATPES, also referred to as an XTEN linker. In some embodiments, the linker is 24 amino acids long. In some embodiments, the linker comprises the amino acid sequence SGGSSGGSSGSETPGTSESATPES. In some embodiments, the linker is 40 amino acids long. In some embodiments, the linker comprises the amino acid sequence SGGSSGGSSGSETPGTSESATPESSGGSSGGSSGGSSGGS. In some embodiments, the linker is 64 amino acids long. In some embodiments, the linker comprises the amino acid sequence SGGSSGGSSGSETPGTSESATPESSGGSSGGSSGGSSGGSSGSETPGTSESATPESSGGSSGGS. In some embodiments, the linker is 92 amino acids in length. In some embodiments, the linker comprises the amino acid sequence

[0126]

[0127] As used in the present disclosure, the term "mutation" refers to the substitution of one residue within a sequence, such as a nucleic acid sequence or an amino acid sequence, with another residue, or the deletion or insertion of one or more residues within a sequence. Typically, a mutation in the present disclosure is described by identifying the original residue, followed by the residue position within the sequence, and by the identity of the newly replaced residue. Various methods for making amino acid substitutions (mutations) provided herein are well known in the art and are provided by, for example, Green and Sambrook, Molecular Cloning: A Laboratory Manual (4th ed., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY (2012)). In some embodiments, the base editors disclosed herein can effectively generate "planned mutations", such as point mutations, in nucleic acids (e.g., nucleic acids within the genome of an individual) without generating a significant number of unplanned mutations, such as unplanned point mutations. In some embodiments, the intended mutation is a mutation generated by a specific base editor (e.g., a cytidine base editor or an adenosine base editor) bound to a guide polynucleotide (e.g., a gRNA) that is specifically designed to generate the intended mutation. Typically, mutations made or identified in a sequence (e.g., an amino acid sequence as described herein) are numbered relative to a reference (or wild-type) sequence (i.e., a sequence that does not contain the mutation). Those skilled in the art readily understand how to determine the position of a mutation in an amino acid sequence and a nucleic acid sequence relative to a reference sequence.

[0128] The term "non-conservative mutation" refers to amino acid substitutions of different groups, for example, lysine replaces tryptophan, or phenylalanine replaces serine, etc. In this case, non-conservative amino acid substitutions are preferred so as not to interfere with or inhibit the biological activity of the functional variant. Non-conservative amino acid substitutions can enhance the biological activity of the functional variant, making the biological activity of the functional variant higher than that of the wild-type protein.

[0129] The term "nuclear localization sequence", "nuclear localization signal", or "NLS" refers to an amino acid sequence that promotes the import of proteins into the nucleus. Nuclear localization sequences are known in the industry and are described, for example, in Plank et al. International PCT Application PCT / EP2000 / 011690, filed November 23, 2000, published as WO / 2001 / 038547 on May 31, 2001, the contents of which are hereby incorporated by reference for the disclosed embodiments of nuclear localization sequences. In other embodiments, the NLS is an optimized NLS such as described by Koblan et al., Nature Biotech. 2018 doi: 10.1038 / nbt.4172. In some embodiments, the NLS comprises the amino acid sequence KRTADGSEFESPKKKRKV, KRPAATKKAGQAKKKK, KKTELQTTNAENKTKKL, KRGINDRNFWRGENGRKTR, RKSGKIAAIVVKRPRK, PKKKRKV, or MDSLLMNRRKFLYQFKNVRWAKGRRETYLC.

[0130] As used in this disclosure, the terms "nucleic acid" and "nucleic acid molecule" refer to a compound comprising a nucleoside base and an acidic moiety, such as a nucleoside, a nucleotide, or a polymer of nucleotides. Typically, a polymeric nucleic acid, for example, a nucleic acid molecule comprising three or more nucleotides, is a linear molecule in which adjacent nucleotides are linked to each other by phosphodiester linkages. In some embodiments, "nucleic acid" refers to individual nucleic acid residues (e.g., nucleotides and / or nucleosides). In some embodiments, "nucleic acid" refers to an oligonucleotide chain comprising three or more individual nucleotide residues. As used in this disclosure, the terms "oligonucleotide", "polynucleotide", and "polynucleic acid" are used interchangeably to refer to a polymer of nucleotides (e.g., a string of at least three nucleotides). In some embodiments, "nucleic acid" encompasses RNA and single-stranded DNA and / or double-stranded DNA. Nucleic acids can occur naturally, for example, in genomes, transcripts, mRNA, tRNA, rRNA, siRNA, snRNA, plastids, cohesin, chromosomes, chromatids, or other naturally occurring nucleic acid molecules. On the other hand, the nucleic acid molecule can be a non-natural molecule, for example, a recombinant DNA or RNA, an artificial chromosome, a genetically modified genome, or a fragment thereof, or a synthetic DNA, RNA, a DNA / RNA hybrid, or comprising non-natural nucleotides or nucleosides. Again, the terms "nucleic acid", "DNA", "RNA" and / or similar terms include nucleic acid analogs, for example, analogs having a backbone other than a phosphodiester. Nucleic acids can be purified from natural sources, produced using a recombinant expression system and optionally, purified, chemically synthesized, etc. When appropriate, for example, in the case of chemically synthesized molecules, nucleic acids can include nucleoside analogs, such as analogs having chemically modified bases or sugars and backbone modifications. Unless otherwise indicated, nucleic acid molecules are presented in a 5' to 3' direction. In some embodiments, the nucleic acid is or comprises a natural nucleoside (e.g., adenosine, thymidine, guanosine, cytidine, uridine, deoxyadenosine, deoxythymidine, deoxyguanosine, and deoxycytidine); a nucleoside analog (e.g., 2-aminoadenosine, 2-thiathymidine, inosine, pyrrolopyrimidine, 3-methyladenosine, 5-methylcytidine, 2-aminoadenosine, C5-bromouridine, C5-fluorouridine, C5-iodouridine, C5-propynyluridine, C5-propynylcytidine, C5-methylcytidine, 2-aminoadenosine, 7-deazaadenosine, 7-deazaguanosine, 8-oxoadenosine, 8-oxoguanosine, 0 6-methylguanine, and 2-thiacytidine); chemically modified bases; biologically modified bases (e.g., methylated bases); inlaid bases, modified sugars (e.g., 2'-fluororibose, ribose, 2'-deoxyribose, arabinose, and hexose); and / or modified phosphate groups (e.g., phosphorothioate and 5'-N-phosphoamidite linkages). In some embodiments, the RNA is an RNA associated with the Cas9 system. For example, the RNA can be a CRISPR RNA (crRNA), a trans-acting small molecule RNA (tracrRNA), a single guide RNA (sgRNA), or a guide RNA (gRNA).

[0131] The terms "nucleobase," "nitrogenous base," or "base" are used interchangeably in this disclosure to refer to nitrogen-containing biological compounds that generate nucleosides, which in turn are constituents of nucleotides. Nucleobases have the ability to form base pairs and to stack with one another, resulting in long helical structures such as ribonucleic acid (RNA) and deoxyribonucleic acid (DNA). Five nucleobases, adenine (A), cytosine (C), guanine (G), thymine (T), and uracil (U), are referred to as primary or canonical. Adenine and guanine are derived from purines; cytosine, uracil, and thymine are derived from pyrimidines. DNA and RNA may also contain other (non-original) modified bases. Non-limiting examples of modified bases include hypoxanthine, xanthine, 7-methylguanine, 5,6-dihydrouracil, 5-methylcytidine (m5C), and 5-hydromethylcytidine. Hypoxanthine and xanthine can be generated through the presence of mutagen, both by deamination (replacing an amino group with a carbonyl group). Hypoxanthine can be modified from adenine. Xanthine can be modified from guanine. Uracil can be obtained from the deamination of cytosine. "Nucleoside" consists of a nucleoside base and a pentose (ribose or deoxyribose). Examples of nucleosides include adenosine, guanosine, uridine, cytidine, 5-methyluridine (m5U), deoxyadenosine, deoxyguanosine, thymidine, deoxyuridine, and deoxycytidine. Examples of nucleosides with modified nucleoside bases include inosine (I), xanthine (X), 7-methylguanosine (m7G), dihydrouridine (D), 5-methylcytidine (m5C), and pseudouridine (Ψ). "Nucleotide" consists of a nucleoside base, a pentose (ribose or deoxyribose), and at least one phosphate group.

[0132] The term "nucleic acid programmable DNA binding protein" or "napDNAbp" can be used interchangeably with "polynucleotide programmable nucleotide binding domain" to refer to a protein associated with a nucleic acid (e.g., DNA or RNA), such as a guide nucleic acid, which guides the napDNAbp to a specific nucleic acid sequence. For example, a Cas9 protein can be associated with a guide RNA, which guides the Cas9 protein to a specific DNA sequence complementary to the guide RNA. In some embodiments, the napDNAbp is a Cas9 domain, for example, a nuclease-active Cas9, a Cas9 nickase (nCas9), or a nuclease-inactive Cas9 (dCas9). Examples of nucleic acid programmable DNA binding proteins include, but are not limited to, Cas9 (e.g., dCas9 and nCas9), Cas12a / Cpf1, Cas12b / C2cl, Cas12c / C2c3, Cas12d / CasY, Cas12e / CasX, Cas12g, Cas12h, and Cas12i. Other nucleic acid programmable DNA binding proteins also fall within the scope of the present disclosure, but may not be specifically listed in the present disclosure. For example, refer to Makarova et al. "Classification and Nomenclature of CRISPR-Cas Systems: Where from Here?" CRISPR J. 2018 Oct; 1: 325-336. doi: 10.1089 / crispr.2018.0033; Yan et al., "Functionally diverse type V CRISPR-Cas systems" Science. 2019 Jan 4; 363(6422): 88-91. doi: 10.1126 / science.aav7271, each of which is incorporated by reference into the disclosure of the present specification.

[0133] As used in the present disclosure, the term "nucleobase editing domain" or "nucleobase editing protein" refers to a protein or enzyme that can catalyze nucleobase modifications in RNA or DNA, such as deamination of cytosine (or cytidine) to uracil (or uridine) or thymine (or thymus), or adenine (or adenosine) to hypoxanthine (or inosine), as well as non-templated nucleotide additions and insertions. In some embodiments, the nucleobase editing domain is a deaminase domain (e.g., cytidine deaminase, cytosine deaminase, adenine deaminase, or adenosine deaminase). In some embodiments, the nucleobase editing domain can be a natural nucleobase editing domain. In some embodiments, the nucleobase editing domain can be a nucleobase editing domain genetically modified or evolved from a natural nucleobase editing domain. The nucleobase editing domain can be obtained from any organism, such as bacteria, humans, chimpanzees, orangutans, monkeys, cows, dogs, rats, or mice. For example, nucleoside base editing proteins are described in International PCT Application Nos. PCT / 2017 / 045381 (WO2018 / 027078) and PCT / US2016 / 058344 (WO2017 / 070632), each of which is incorporated by reference into the disclosure of this specification in its entirety. Please also refer to Komor, AC, et al., "Programmable editing of target bases in genomic DNA without double-stranded DNA cleavage," Nature 533, 420-424 (2016); Gaudelli, NM, et al., "Programmable base editing of A·T to G·C in genomic DNA without DNA cleavage," Nature 551, 464-471 (2017); and Komor, AC, et al., "Improved base excision repair inhibition and bacteriophage μGam protein with higher efficiency and product purity to obtain C:G-to-T:A base editor," Science Advances 3:eaao4774 (2017), the entire contents of which are incorporated by reference into the disclosure of this specification.

[0134] As used in this disclosure, "obtaining" as in "obtaining an agent" includes synthesizing, purchasing, or otherwise obtaining the agent.

[0135] As used in this disclosure, "patient" or "subject" refers to a mammalian subject or individual who is diagnosed with, at risk of suffering from or developing a disease or condition, or suspected of suffering from or developing a disease or condition. In some embodiments, the term "patient" refers to a mammalian subject with a higher than average probability of developing a disease or condition. Examples of patients can be humans, non-human primates, cats, dogs, pigs, cows, cats, horses, camels, alpacas, goats, sheep, rodents (e.g., mice, rabbits, rats, or guinea pigs) and other mammals that can benefit from the therapies disclosed herein. Examples of human patients can be men and / or women.

[0136] "Patient in need" or "individual in need" in this disclosure refers to a patient diagnosed with or suspected of having a disease or condition such as, but not limited to, alpha-1 antitrypsin deficiency (A1AD).

[0137] The terms "pathogenic mutation", "pathogenic variant", "disease-causing mutation", "pathogenic variant", "deleterious mutation", or "susceptibility mutation" refer to a genetic alteration or mutation that increases a person's susceptibility to a disease or condition or to an infection. In some embodiments, the pathogenic mutation comprises a substitution of at least one wild-type amino acid with at least one pathogenic amino acid in a protein encoded by a gene.

[0138] The terms "peptide", "polypeptide", "protein", and their grammatical synonyms are used interchangeably in this disclosure and refer to polymers of amino acid residues linked together by peptide (amide) bonds. The term refers to proteins, peptides, or polypeptides of any size, structure, or function. Typically, a protein, peptide, or polypeptide is at least three amino acids long. A protein, peptide, or polypeptide may refer to an individual protein or a collection of proteins. One or more amino acids in a protein, peptide, or polypeptide may be modified, for example, by the addition of chemical entities, such as carbohydrate groups, hydroxyl groups, phosphate groups, farnesyl groups, isofarnesyl groups, fatty acid groups, linking groups for conjugation, functionalization, or other modifications. A protein, peptide, or polypeptide may also be a single molecule, or may be a multimolecular complex. A protein, peptide, or polypeptide may be just a fragment of a natural protein or peptide. A protein, peptide, or polypeptide may be natural, recombinant, or synthetic, or any combination thereof. As used in this disclosure, the term "fusion protein" refers to a hybrid polypeptide comprising protein domains from at least two different proteins. A protein can be located at the amino-terminal (N-terminal) portion or at the carboxyl-terminal (C-terminal) portion of the fusion protein, thereby generating an amino-terminal fusion protein or a carboxyl-terminal fusion protein, respectively. The protein can include different domains, for example, a nucleic acid binding domain (e.g., a gRNA binding domain of Cas9 that directs protein binding to a target site) and a nucleic acid cleavage domain, or a catalytic domain of a nucleic acid editing protein. In some embodiments, the protein includes a protein-containing portion, for example, an amino acid sequence that constitutes a nucleic acid binding domain, and an organic compound, such as a compound that can act as a nucleic acid cleavage agent. In some embodiments, the protein is complexed or associated with a nucleic acid such as RNA or DNA. Any protein provided in the present disclosure can be manufactured by any method known in the industry. For example, the protein provided in the present disclosure can be manufactured by recombinant protein expression and purification, which is particularly suitable for fusion proteins comprising a peptide linker. Recombinant protein expression and purification methods are well known, including those described by Green and Sambrook, Molecular Cloning: A Laboratory Manual (4th ed., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY (2012)), the disclosure of which is incorporated herein by reference in its entirety.

[0139] The polypeptides and proteins disclosed herein (including functional proteins and functional variants thereof) may contain synthetic amino acids replacing one or more natural amino acids. Such amino acids are known in the industry and include homoserine, S-acetylaminomethyl-cysteine, trans-3- and trans-4-hydroxyproline, 4-aminophenylalanine, 4-nitrophenylalanine, 4-chlorophenylalanine, 4-carboxyphenylalanine, β-phenylserine, β-hydroxyphenylalanine, phenylglycine, α-naphthylalanine, cyclohexylalanine, cyclohexylglycine, indoline-2-carboxylic acid, 1,2,3,4-tetrahydroisoquinoline ... In some embodiments, the present invention relates to quinoline-3-carboxylic acid, aminomalonic acid, aminomalonic acid monoamide, N'-benzyl-N'-methyl-lysine, N',N'-dibenzyl-lysine, 6-hydroxylysine, ornithine, α-aminocyclopentanecarboxylic acid, α-aminocyclohexanecarboxylic acid, α-aminocycloheptanecarboxylic acid, α-(2-amino-2-norbornane)-carboxylic acid, α,γ-diaminobutyric acid, α,β-diaminopropionic acid, homophenylalanine, and α-tert-butylglycine. Polypeptides and proteins may be associated with post-translational modifications of one or more amino acids of the polypeptide construct. Non-limiting examples of post-translational modifications include phosphorylation, acylation including acetylation and formylation, glycosylation (including N-linked and O-linked), amidation, hydroxylation, alkylation including methylation and ethylation, ubiquitination, addition of pyrrolidonecarboxylic acid, formation of disulfide bridges, sulfation, myristoylation, palmitoylation, prenylation, farnesylation, geranylation, glycosylphosphatidylinositolization, lipidation, and iodination.

[0140] The term "polynucleotide programmable nucleotide binding domain" refers to a protein associated with a nucleic acid (e.g., DNA or RNA), such as a guide polynucleotide (e.g., a guide RNA), which guides the polynucleotide programmable DNA binding domain to a specific nucleic acid sequence. In some embodiments, the polynucleotide programmable nucleotide binding domain is a polynucleotide programmable DNA binding domain. In some embodiments, the polynucleotide programmable nucleotide binding domain is a polynucleotide programmable RNA binding domain. In some embodiments, the polynucleotide programmable nucleotide binding domain is a Cas9 protein. The Cas9 protein can be associated with a guide RNA that guides the Cas9 protein to a specific DNA sequence that is complementary to the guide RNA. In some embodiments, the polynucleotide programmable nucleotide binding domain has a Cas9 domain, for example, a nuclease-active Cas9, a Cas9 nickase (nCas9), or a nuclease-inactive Cas9 (dCas9). Non-limiting examples of nucleic acid programmable DNA binding proteins include Cas9 (e.g., dCas9 and nCas9), Cas12a / Cpf1, Cas12b / C2cl, Cas12c / C2c3, Cas12d / CasY, Cas12e / CasX, Cas12g, Cas12h, and Cas12i. Non-limiting examples of Cas include Cas1, Cas1B, Cas2, Cas3, Cas4, Cas5, Cas5d, Cas5t, Cas5h, Cas5a, Cas6, Cas7, Cas8, Cas8a, Cas8b, Cas8c, Cas9 (also known as Csn1 or Csx12), Cas10, Cas10d, Cas12a / Cpf1, Cas12b / C2cl, Cas12c / C2c3, Cas12d / CasY, Cas12e / CasX, Cas12g, Cas12h, Cas12i, Csy1, Csy2, Csy3, Csy4, Cse1, Cse2, Cse3, Cse4, Cse5e, Csc1, Csc2, Csa 5. Csn1, Csn2, Csm1, Csm2, Csm3, Csm4, Csm5, Csm6, Cmr1, Cmr3, Cmr4, Cmr5, Cmr6, Csb1, Csb2, Csb3, Csx17, Csx14, Csx10, Csx16, CsaX, Csx3, Csx1, Csx1S, Csx11, Csf1, Csf2, CsO, Csf4, Csd1, Csd2, Cst1, Cst2, Csh1, Csh2, Csa1, Csa2, Csa3, Csa4, Csa5, type II Cas effector protein, type V Cas effector protein, type VI Cas effector protein, CARF, DinG, their equivalents, or modified or engineered versions thereof.Other nucleic acid programmable DNA binding proteins also fall within the scope of the present disclosure but are not specifically listed herein.

[0141] As used in the context of proteins or nucleic acids in this disclosure, the term "recombinant" refers to proteins or nucleic acids that do not occur in nature but are the product of human engineering. For example, in some embodiments, a recombinant protein or nucleic acid molecule comprises an amino acid or nucleotide sequence that contains at least one, at least two, at least three, at least four, at least five, at least six, or at least seven mutations compared to any native sequence.

[0142] "Decrease" means a negative change of at least 10%, 25%, 50%, 75%, or 100%.

[0143] "Reference" refers to standard or control conditions. In one embodiment, the reference is a wild-type or healthy cell.

[0144] "Reference sequence" means a defined sequence used as a basis for sequence comparison. A reference sequence can be a subset or the entirety of a particular sequence, for example, a segment of a full-length cDNA or gene sequence, or a complete cDNA or gene sequence. For polypeptides, the length of a reference polypeptide sequence is typically at least about 16 amino acids, preferably at least about 20 amino acids, more preferably at least about 25 amino acids, and even more preferably about 35 amino acids, about 50 amino acids, or about 100 amino acids. For nucleic acids, the length of a reference nucleic acid sequence is typically at least about 50 nucleotides, preferably at least about 60 nucleotides, more preferably at least about 75 nucleotides, and even more preferably about 100 nucleotides, or about 300 nucleotides, or any integers therearound or therebetween.

[0145] The terms "RNA programmable nuclease" and "RNA guided nuclease" are used with (e.g., bound to or associated with) one or more RNAs that are not targets for cleavage. In some embodiments, the RNA programmable nuclease, when complexed with RNA, can be referred to as a nuclease:RNA complex. Typically, the bound RNA is referred to as a guide RNA (gRNA). The gRNA can exist as a complex of two or more RNAs, or as a single RNA molecule. A gRNA that exists as a single RNA molecule can be referred to as a single guide RNA (RNA), but "gRNA" is used interchangeably to refer to a guide RNA that exists as a single molecule or as a complex of two or more RNAs. Typically, a gRNA that exists as a single RNA comprises two domains: (1) a domain that shares homology with a target nucleic acid (e.g., and guides the Cas9 complex to bind to the target); and (2) a domain that binds to the Cas9 protein. In some embodiments, domain (2) corresponds to a sequence called tracrRNA and comprises a stem-loop structure. For example, in some embodiments, domain (2) is the same as or homologous to tracrRNA, as provided in Jinek et ah, Science 337:816-821 (2012), which is incorporated herein by reference in its entirety. Other examples of gRNA (e.g., including domain 2) are described in U.S. Provisional Patent Application No. USSN 61 / 874,682, filed on September 6, 2013, entitled "Switchable Cas9 Nucleases And Uses Thereof," and U.S. Provisional Patent Application No. USSN 61 / 874,746, filed on September 6, 2013, entitled "Delivery System For Functional Nucleases," which are incorporated herein by reference in their entirety. In some embodiments, a gRNA comprises two or more of domains (1) and (2), which may be referred to as an "extended gRNA." For example, as described herein, the extended gRNA will, for example, bind two or more Cas9 proteins and bind to a target nucleic acid at two or more separate regions. The gRNA comprises a nucleotide sequence complementary to the target site, which mediates the binding of the nuclease / RNA complex to the target site, providing sequence specificity of the nuclease: RNA complex.In some embodiments, the RNA programmable nuclease is a (CRISPR-associated system) Cas9 endonuclease, such as Cas9 from Streptococcus pyogenes (Csn1) (e.g., see "Complete genome sequence of an M1 strain of Streptococcus pyogenes." Ferretti JJ, McShan WM, Ajdic DJ, Savic DJ, Savic G., Lyon K., Primeaux C, Sezate S., Suvorov AN, Kenton S., Lai HS, Lin SP, Qian Y., Jia HG, Najar F.Z., Ren Q., Zhu H., Song L., White J., Yuan X., Clifton SW, Roe BA, McLaughlin RE, Proc. Natl. Acad. Sci. USA 98: 4658-4663 (2001); "CRISPR RNA maturation by trans-encoded small RNA and host factor RNase III." Deltcheva E., Chylinski K., Sharma CM., Gonzales K., Chao Y., Pirzada ZA, Eckert MR, Vogel J., Charpentier E., Nature 471: 602-607 (2011).

[0146] "SERPINA1 polynucleotide" refers to a nucleic acid molecule encoding an A1AT protein or a fragment thereof. An example sequence of a SERPINA1 polynucleotide available under NCBI Accession No. NM_000295 is provided below:

[0147]

[0148]

[0149]

[0150] The base positions complementary to the PAM sequence are shown in italics and double underlining. The G at position 1455, which is complementary to the target C at position 1455, is indicated in bold and underlined.

[0151] The term "single nucleotide polymorphism (SNP)" refers to a single nucleotide variation that occurs at a specific position in a genome, where each variation is present to a certain appreciable degree within a population (e.g., >1%). For example, at a specific base position in a human genome, a C nucleotide may occur in most individuals, but in a few individuals, the position is occupied by an A. This indicates that there is a SNP at this specific position, and the two possible nucleotide variations, C or A, are the alleles for this position. SNPs underlie differences in susceptibility to disease (a wide range of human diseases). Disease severity and the body's response to treatment are also indicative of genetic variation. SNPs can fall within the coding region of a gene, the non-coding region of a gene, or the intergenic region (the region between genes). In some embodiments, SNPs within the coding sequence do not necessarily change the amino acid sequence of a protein, which is produced due to the degeneracy of the genetic code. SNPs in the coding region are divided into two types: synonymous SNPs and non-synonymous SNPs. Synonymous SNPs do not affect the protein sequence, while non-synonymous SNPs change the amino acid sequence of a protein. Non-synonymous SNPs are divided into two types: missense and nonsense. SNPs that are not in the protein coding region may still affect gene splicing, transcription factor binding, signaling RNA degradation, or the sequence of coding RNA. Gene expression affected by this type of SNP, called eSNP (expression SNP), can be upstream or downstream of the gene. Single nucleotide variants (SNVs) are variations in a single nucleotide without any frequency restrictions and may occur in somatic cells. Somatic single nucleotide variations (e.g., caused by cancer) are also called single nucleotide changes.

[0152] "Specific binding" refers to a nucleic acid molecule, polypeptide, or a complex thereof (e.g., a nucleic acid programmable DNA binding domain and a guide nucleic acid), compound, or molecule that recognizes and binds to a polypeptide and / or nucleic acid molecule of the invention, but does not substantially recognize and bind to other molecules in a sample (e.g., a biological sample).

[0153] Nucleic acid molecules useful in the methods of the present invention include nucleic acid molecules encoding polypeptides of the present invention or fragments thereof. Such nucleic acid molecules need not be 100% identical to endogenous nucleic acid sequences, but will typically have substantial identity. Polynucleotides having "substantial identity" to endogenous sequences are typically capable of hybridizing with at least one strand of a double-stranded nucleic acid molecule. Nucleic acid molecules useful in the methods of the present invention include nucleic acid molecules encoding polypeptides of the present invention or fragments thereof. Such nucleic acid molecules need not be 100% identical to endogenous nucleic acid sequences, but will typically have substantial identity. Polynucleotides having "substantial identity" to endogenous sequences are typically capable of hybridizing with at least one strand of a double-stranded nucleic acid molecule. "Hybridization" means pairing to generate double-stranded molecules, or portions thereof, between complementary polynucleotide sequences (e.g., genes described herein) under various stringency conditions (e.g., see Wahl, GM and SL Berger (1987) Methods Enzymol. 152: 399; Kimmel, AR (1987) Methods Enzymol. 152: 507).

[0154] For example, stringent salt concentrations will typically be less than about 750 mM NaCl and 75 mM trisodium citrate, preferably less than about 500 mM NaCl and 50 mM trisodium citrate, and more preferably less than about 250 mM NaCl and 25 mM trisodium citrate. Low stringency hybridization can be obtained in the absence of an organic solvent (e.g., formamide), while high stringency hybridization can be obtained in the presence of at least about 35% formamide, and more preferably at least about 50% formamide. Stringent temperature conditions typically include a temperature of at least about 30° C., more preferably at least about 37° C., and most preferably at least about 42° C. Various additional parameters, such as hybridization time, concentration of detergent (e.g., sodium dodecyl sulfate (SDS)), and inclusion or exclusion of carrier DNA, are well known to those skilled in the art. Various degrees of stringency are achieved by combining these various conditions as desired. In a preferred embodiment, hybridization will occur at 30°C in 750 mM NaCl, 75 mM trisodium citrate, and 1% SDS. In a more preferred embodiment, hybridization will occur at 37°C in 500 mM NaCl, 50 mM trisodium citrate, 1% SDS, 35% formamide, and 100 μg / ml denatured salmon sperm (ssDNA). In the most preferred embodiment, hybridization will occur at 42°C in 250 mM NaCl, 25 mM trisodium citrate, 1% SDS, 50% formamide, and 200 μg / ml ssDNA. Useful variations on these conditions will be apparent to those skilled in the art.

[0155] For most applications, the stringency of the washing step after hybridization will also vary. Washing stringency conditions can be defined by salt concentration and by temperature. As described above, washing stringency can be increased by reducing salt concentration and by increasing temperature. For example, the stringent salt concentration of the washing step is preferably less than about 30mM NaCl and 3mM trisodium citrate; and optimally less than about 15mM NaCl and 1.5mM trisodium citrate. Stringent temperatures for the washing step typically include temperatures of at least about 25°C, more preferably at least about 42°C, and even more preferably at least about 68°C. In a preferred embodiment, the washing step will occur at 25°C in 30mM NaCl, 3mM trisodium citrate, and 0.1% SDS. In a more preferred embodiment, the washing step will occur at 42°C in 15mM NaCl, 1.5mM trisodium citrate, and 0.1% SDS. In a more preferred embodiment, the wash step will occur at 68°C in 15 mM NaCl, 1.5 mM trisodium citrate, and 0.1% SDS. Additional variations on these conditions will be apparent to those skilled in the art. Hybridization techniques are well known to those skilled in the art and are described in, for example, Benton and Davis (Science 196:180, 1977); Grunstein and Hogness (Proc. Natl. Acad. Sci., USA 72:3961, 1975); Ausubel et al. (Current Protocols in Molecular Biology, Wiley Interscience, New York, 2001); Berger and Kimmel (Guide to Molecular Cloning Techniques, 1987, Academic Press, New York); and Sambrook et al., Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory Press, New York.

[0156] "Subject" means a mammal, including but not limited to a human or a non-human mammal, such as a cow, horse, dog, sheep, or cat.

[0157] "Substantially identical" refers to a polypeptide or nucleic acid molecule that is at least 50% identical to a reference amino acid sequence (e.g., any of the amino acid sequences described herein) or nucleic acid sequence (e.g., any of the nucleic acid sequences described herein). Preferably, such a sequence is at least 60% identical at the amino acid level or nucleic acid to the sequence used for comparison, more preferably 80% or 85%, and more preferably 90%, 95%, or even 99% identical.

[0158] Sequence identity is typically determined using sequence analysis software (e.g., Sequence Analysis Software Package of the Genetics Computer Group, University of Wisconsin Technical Center, 1710 University Avenue, Madison, Wis. 53705, BLAST, BESTFIT, GAP, or PILEUP / PRETTYBOX programs). Such software matches identical or similar sequences by assigning degrees of homology to each substitution, deletion, and / or other modification. Conservative substitutions typically include substitutions within the following groups: glycine, alanine, valine, isoleucine, leucine; asparagine, glutamine; asparagine, glutamic acid; serine, threonine; lysine, arginine; and phenylalanine, tyrosine. In an embodiment of a method for determining identity, a BLAST program may be used, with e -3 To e -100 The probability fraction between them indicates closely related sequences.

[0159] The term "target site" refers to a sequence within a nucleic acid molecule that is modified by a nucleoside base editor. In one embodiment, the target site is deaminated by a deaminase or a fusion protein comprising a deaminase (eg, cytidine deaminase or adenosine deaminase).

[0160] Because RNA-programmable nucleases (eg, Cas9) use RNA:DNA hybridization to target DNA cleavage sites, in principle, these proteins can be targeted to any sequence specified by a guide RNA. The use of RNA programmable nucleases, such as Cas9, for site-specific cleavage (e.g., to modify the genome) is known in the art (e.g., see Cong, L. et ah, Multiplex genome engineering using CRISPR / Cas systems. Science 339, 819-823 (2013); Mali, P. et ah, RNA-guided human genome engineering via Cas9. Science 339, 823-826 (2013); Hwang, W. et ah, Efficient genome editing in zebrafish using a CRISPR-Cas system. Nature biotechnology 31, 227-229 (2013); Jinek, M. et ah, RNA-programmed genome editing in human cells. eLife 2, e00471 (2013); Dicarlo, JE et ah, Genome engineering in Saccharomyces cerevisiae using CRISPR-Cas system. Nucleic acids research (2013); Jiang, W. et ah RNA-guided editing of bacterial genomes using CRISPR-Cas systems. Nature biotechnology 31, 233-239 (2013); the entire texts of each of which are incorporated herein by reference).

[0161] As used in this disclosure, the terms "therapy," "treatment," or grammatical synonyms thereof refer to obtaining a desired pharmacological and / or physiological effect. In some embodiments, the effect is therapeutic, that is, the effect partially or completely cures a disease and / or the adverse symptoms attributable to the disease. In some embodiments, the effect is prophylactic, that is, the effect prevents the occurrence or recurrence of a disease or condition. To achieve this goal, the disclosed methods comprise administering a therapeutically effective amount of a composition as described herein.

[0162] "Uracil glycosylase inhibitor" refers to an inhibitor of the uracil excision repair system. In one embodiment, the inhibitor is a protein or fragment thereof that binds to the host uracil-DNA glycosylase and prevents the removal of uracil residues from DNA.

[0163] It should be understood that the ranges provided in this disclosure are shorthand for all values ​​within the range. For example, it should be understood that a range of 1 to 50 includes any number, combination of numbers, or sub-range from the group consisting of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, or 50.

[0164] In any definition of a variable in the present disclosure, a list of chemical groups includes definitions of the variable as any single group or combination of listed groups. A reference to an embodiment of a variable or aspect in the present disclosure includes that embodiment as a single embodiment or in combination with any other embodiment or portion thereof.

[0165] Any composition or method provided in this disclosure can be combined with one or more of any other composition or method provided in this disclosure.

[0166] DNA editing has become a viable approach to modify disease states by correcting pathogenic mutations at the genomic level. Until recently, all DNA editing platforms functioned by inducing DNA double-strand breaks (DSBs) at specific genomic loci and relying on endogenous DNA repair pathways to determine product outcomes in a semi-stochastic manner, resulting in a complex population of gene products. Although precise user-defined repair outcomes can be achieved through the homology-directed repair (HDR) pathway, there are many challenges that prevent the use of HDR's efficient repair in therapeutically relevant cell types. In fact, this pathway is less effective than the competing error-prone non-homologous end joining pathway. In addition, HDR is highly restricted to the G1 and S phases of the cell cycle, hindering the precise repair of DSBs in post-mitotic cells. As a result, it has been proven difficult or impossible to alter genomic sequences in a user-defined programmable manner in these populations.

[0167] Nucleoside Base Editor

[0168] Disclosed herein is a base editor or nucleoside base editor for editing, modifying, or changing a target nucleotide sequence of a polynucleotide. Described herein is a nucleoside base editor or base editor comprising a polynucleotide programmable nucleotide binding domain or a nucleoside base editing domain. The polynucleotide programmable nucleotide binding domain, when combined with a bound guide polynucleotide (e.g., gRNA), can specifically bind to a target polynucleotide sequence (in other words, by complementary base pairing between the bases of the bound guide nucleic acid and the bases of the target polynucleotide sequence), thereby positioning the base editor to the target nucleic acid sequence desired to be edited. In some embodiments, the target polynucleotide sequence comprises single-stranded DNA or double-stranded DNA. In some embodiments, the target polynucleotide sequence comprises RNA. In some embodiments, the target polynucleotide sequence comprises a DNA-RNA fusion.

[0169] Polynucleotide programmable nucleotide binding domain

[0170] The term "polynucleotide programmable nucleotide binding domain" refers to a protein associated with a nucleic acid (e.g., DNA or RNA), such as a guide polynucleotide (e.g., a guide RNA), which guides the polynucleotide programmable nucleotide binding domain to a specific nucleic acid sequence. In some embodiments, the polynucleotide programmable nucleotide binding domain is a polynucleotide programmable DNA binding domain. In some embodiments, the polynucleotide programmable nucleotide binding domain is a polynucleotide programmable RNA binding domain. In some embodiments, the polynucleotide programmable nucleotide binding domain is a Cas9 protein. In some embodiments, the polynucleotide programmable nucleotide binding domain is a Cpf1 protein.

[0171] CRISPR is an adaptive immune system that provides protection against mobile genetic elements (viruses, replaceable elements, and conjugative plastids). CRISPR clusters contain spacers, complementary sequences of ancestral mobile elements, and target invading nucleic acids. CRISPR clusters are transcribed and processed into CRISPR RNA (crRNA). In type II CRISPR systems, the correct processing of pre-crRNA requires transcoding small molecule RNA (tracrRNA), endogenous ribonuclease 3 (rnc), and Cas9 protein. TracrRNA is used as a guide for ribonuclease 3-assisted processing of pre-crRNA. Subsequently, Cas9 / crRNA / tracrRNA endonucleases cleave linear or circular dsDNA targets complementary to the spacer. Target strands that are not complementary to crRNA are first cut endonucleases and then trimmed 3'-5' by exonucleases. In nature, DNA binding and DNA cleavage typically require proteins and two RNAs. However, a single guide RNA ("sgRNA" or simply "gRNA") can be genetically engineered to combine the aspects of both crRNA and tracrRNA into a single RNA. For example, see Jinek M., Chylinski K., Fonfara I., Hauer M., Doudna JA, Charpentier E. Science 337:816-821 (2012), which is incorporated herein by reference in its entirety. Cas9 recognizes short motifs (PAM or protospacer adjacent motifs) in CRISPR repeat sequences to help distinguish self from non-self.

[0172] The sequence and structure of the Cas9 nuclease are well known to those skilled in the art (e.g., see “Complete genome sequence of Streptococcus pyogenes M1 strain” Ferretti JJ, McShan WM, Ajdic DJ, Savic DJ, Savic G., Lyon K., Primeaux C, Sezate S., Suvorov AN, Kenton S., Lai HS, Lin SP, Qian Y., Jia HG, Najar FZ, Ren Q., Zhu H., Song L., White J., Yuan X., Clifton SW, Roe BA, McLaughlin RE, Proc. Natl. Acad. Sci. USA 98:4658-4663 (2001); “CRISPR RNA maturation by transcoding small RNA and host factor RNase III” Deltcheva E., Chylinski K., Sharma CM., Gonzales K., Chao Y., Pirzada ZA, Eckert MR, Vogel J., Charpentier E., Nature 471:602-607 (2011); and "A programmable dual-RNA-guided DNA endonuclease in adaptive bacterial immunity." Jinek M., Chylinski K., Fonfara I., Hauer M., Doudna JA, Charpentier E. Science 337:816-821 (2012), each of which is incorporated herein by reference in its entirety). Cas9 homologous genes have been described in various species, including but not limited to Streptococcus pyogenes and Streptococcus thermophilus.Based on the disclosure herein, additional suitable Cas9 nucleases and sequences are readily apparent to those skilled in the art, including Cas9 sequences and loci obtained from organisms, which are disclosed in Chylinski, Rhun, and Charpentier, "The tracrRNA and Cas9 families of type II CRISPR-Cas immunity systems" (2013) RNA Biology 10:5, 726-737, which is incorporated herein by reference in its entirety.

[0173] In some aspects, the nucleic acid programmable DNA binding protein (napDNAbp) is a Cas9 domain. Non-limiting examples of Cas9 domains are provided in the present disclosure. The Cas9 domain can be a nuclease active Cas9 domain, a nuclease inactive Cas9 domain, or a Cas9 nickase. In some embodiments, the Cas9 domain is a nuclease active domain. For example, the Cas9 domain can be a Cas9 domain that cuts two strands of a diploid nucleic acid (two strands of a diploid DNA molecule). In some embodiments, the Cas9 domain comprises any one of the amino acid sequences as described in the present disclosure. In some embodiments, the Cas9 domain comprises an amino acid sequence that is at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or at least 99.5% identical to any one of the amino acid sequences as described in the present disclosure. In some embodiments, the Cas9 domain comprises an amino acid sequence having 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 21, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50 or more mutations compared to any of the amino acid sequences set forth in the disclosure. In some embodiments, the Cas9 domain comprises an amino acid sequence having at least 10, at least 15, at least 20, at least 30, at least 40, at least 50, at least 60, at least 70, at least 80, at least 90, at least 100, at least 150, at least 200, at least 250, at least 300, at least 350, at least 400, at least 500, at least 600, at least 700, at least 800, at least 900, at least 1000, at least 1100, or at least 1200 identical adjacent amino acid residues compared to any of the amino acid sequences set forth in the present disclosure.

[0174] In some embodiments, the Cas9 nuclease has an inactive (e.g., inactivated) DNA cleavage domain, in other words, Cas9 is a nickase. Nuclease-inactivated Cas9 proteins are interchangeably referred to as "dCas9" proteins (for nuclease-dead Cas9). Methods for generating Cas9 proteins (or fragments thereof) with inactive DNA cleavage domains are known (e.g., see Jinek et al, Science. 337: 816-821 (2012); Qi et al, "Repurposing CRISPR as an RNA-Guided Platform for Sequence-Specific Control of Gene Expression" (2013) Cell. 28; 152 (5): 1173-83, each of which is incorporated herein by reference in its entirety). For example, the DNA cleavage domain of Cas9 is known to include two subdomains, the HNH nuclease subdomain and the RuvC1 subdomain. The HNH subdomain cleaves the complementary strand of the gRNA, while the RuvC1 subdomain cleaves the non-complementary strand. Mutations within these subdomains can suppress the nuclease activity of Cas9. For example, mutations D10A and H840A completely inactivate the nuclease activity of Streptococcus pyogenes Cas9 (Jinek et al, Science. 337: 816-821 (2012); Qi et al, Cell. 28; 152 (5): 1173-83 (2013)). In some embodiments, the Cas9 nuclease has an inactivated (e.g., inactivated) DNA cleavage domain, in other words, Cas9 is a nickase, referred to as a "nCas9" protein (for "nickase" Cas9). In some embodiments, a protein comprising a fragment of Cas9 is provided. For example, in some embodiments, the protein comprises one of the two Cas9 domains: (1) the gRNA binding domain of Cas9; or (2) the DNA cleavage domain of Cas9. In some embodiments, a protein comprising Cas9 or a fragment thereof is referred to as a "Cas9 variant". A Cas9 variant shares homology with Cas9 or a fragment thereof. For example, a Cas9 variant is at least about 70% identical, at least about 80% identical, at least about 90% identical, at least about 95% identical, at least about 96% identical, at least about 97% identical, at least about 98% identical, at least about 99% identical, at least about 99.5% identical, or at least about 99.9% identical to wild-type Cas9.In some embodiments, the Cas9 variants may have 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 21, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50 or more amino acid changes compared to wild-type Cas9. In some embodiments, the Cas9 variant comprises a fragment of Cas9 (e.g., a gRNA binding domain or a DNA cleavage domain) such that the fragment is at least about 70% identical, at least about 80% identical, at least about 90% identical, at least about 95% identical, at least about 96% identical, at least about 97% identical, at least about 98% identical, at least about 99% identical, at least about 99.5% identical, or at least about 99.9% identical to the corresponding fragment of wild-type Cas9. In some embodiments, the fragment is at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95% identical, at least 96%, at least 97%, at least 98%, at least 99%, or at least 99.5% identical to the corresponding wild-type Cas9 in amino acid length. In some embodiments, the fragment is at least 100 amino acids long. In some embodiments, the fragment is at least 100, 150, 200, 250, 300, 350, 400, 450, 500, 550, 600, 650, 700, 750, 800, 850, 900, 950, 1000, 1050, 1100, 1150, 1200, 1250, or at least 1300 amino acids in length.

[0175] In some embodiments, the wild-type Cas9 corresponds to Cas9 obtained from Streptococcus pyogenes (NCBI Reference Sequence: NC_017053.1, nucleotide sequence and amino acid sequence are as follows).

[0176]

[0177]

[0178]

[0179] (Single lower line: HNH domain; double lower line: RuvC domain)

[0180] In some embodiments, the wild-type Cas9 corresponds to or comprises the following nucleotide and / or amino acid sequence:

[0181]

[0182]

[0183] (Single lower line: HNH domain; double lower line: RuvC domain)

[0184] In some embodiments, the wild-type Cas9 corresponds to Cas9 obtained from Streptococcus pyogenes (NCBI reference sequence: NC_002737.2 (nucleotide sequence as follows); and Uniprot reference sequence: Q99ZW2 (amino acid sequence as follows).

[0185]

[0186]

[0187] (Single lower line: HNH domain; double lower line: RuvC domain)

[0188] In some embodiments, Cas9 refers to Cas9 obtained from Corynebacterium ulcerans (NCBI Refs: NC_015683.1, NC_017317.1); Corynebacterium diphtheria (NCBI Refs: NC_016782.1, NC_016786.1); Spiroplasma syrphidicola (NCBI Ref: NC_021284.1); Prevotella intermedia (NCBI Ref: NC_017861.1); Spiroplasma taiwanense (NCBI Ref: NC_021846.1); Streptococcus iniae (NCBI Ref: NC_021847.1); Ref: NC_021314.1); Belliella baltica (NCBI Ref: NC_018010.1); Psychroflexus torquis I (NCBI Ref: NC_018721.1); Streptococcus thermophilus (NCBI Ref: YP_820832.1); Listeria innocua (NCBI Ref: NP_472073.1); Campylobacter jejuni (NCBI Ref: YP_002344900.1); or Neisseria meningitidis (NCBI Ref: YP_002342100.1), or refers to Cas9 obtained from any other organism.

[0189] In some embodiments, dCas9 corresponds to, or partially or completely comprises, a Cas9 amino acid sequence having one or more mutations that inactivate Cas9 nuclease activity. Unless otherwise stated, Cas9 is labeled relative to a wild-type reference sequence. For example, in some embodiments, a dCas9 domain comprises D10A and H840A mutations, or corresponding mutations in another Cas9. In some embodiments, a dCas9 domain comprises the amino acid sequence of dCas9 (D10A and H840A).

[0190] (Single lower line: HNH domain; double lower line: RuvC domain)

[0191] In several embodiments, the Cas9 domain comprises a D10A mutation, while the residue at position 840 remains a histidine in the amino acid sequence provided above, or a corresponding position in any of the amino acid sequences provided in the present disclosure.

[0192] In other embodiments, dCas9 variants having mutations other than D10A and H840A are provided, for example, resulting in nuclease-inactivated Cas9 (dCas9). For example, such mutations include other amino acid substitutions at D10A and H840A, or other substitutions in the Cas9 nuclease domain (e.g., substitutions in the HNH nuclease subdomain and / or the RuvC1 subdomain). In some embodiments, variants or homologs of dCas9 are provided that are at least about 70% identical, at least about 80% identical, at least about 90% identical, at least about 95% identical, at least about 98% identical, at least about 99% identical, at least about 99.5% identical, or at least about 99.9% identical. In some embodiments, dCas9 variants having shorter or longer amino acid sequences, up to about 5 amino acids, up to about 10 amino acids, up to about 15 amino acids, up to about 20 amino acids, up to about 25 amino acids, up to about 30 amino acids, up to about 40 amino acids, up to about 50 amino acids, up to about 75 amino acids, up to about 100 amino acids are provided.

[0193] In some embodiments, a Cas9 fusion protein as provided in the present disclosure comprises the full-length amino acid sequence of a Cas9 protein, e.g., one of the Cas9 sequences provided in the present disclosure. However, in other embodiments, a fusion protein as provided in the present disclosure does not comprise the full-length Cas9 sequence, but instead comprises only one or more Cas9 fragments. Examples of amino acid sequences of suitable Cas9 domains and Cas9 fragments are provided in the present disclosure, and the sequences of other suitable Cas9 domains and Cas9 fragments will be apparent to those skilled in the art.

[0194] The Cas9 protein can be associated with a guide RNA that guides the Cas9 protein to a specific DNA sequence that is complementary to the guide RNA. In some embodiments, the polynucleotide programmable nucleotide binding domain is a Cas9 domain, e.g., a nuclease-active Cas9, a Cas9 nickase (nCas9), or a nuclease-inactive Cas9 (dCas9). Examples of nucleic acid programmable DNA binding proteins include, but are not limited to, Cas9 (e.g., dCas9 and nCas9), CasX, CasY, Cpf1, Cas12b / C2c1, and Cas12c / C2c3.

[0195] Nuclease-inactivated Cas9 proteins are interchangeably referred to as "dCas9" proteins (for nuclease-"dead" Cas9) or catalytically inactivated Cas9. Methods for generating Cas9 proteins (or fragments thereof) with inactivated DNA cleavage domains are known (e.g., see Jinek et al, Science. 337: 816-821 (2012); Qi et al, "Repurposing CRISPR as an RNA-guided platform for specific sequence control of gene expression" (2013) Cell. 28; 152 (5): 1173-83, each of which is incorporated herein by reference in its entirety). For example, the DNA cleavage domain of Cas9 is known to include two subdomains, the HNH nuclease subdomain and the RuvC1 subdomain. The HNH subdomain cleaves the complementary strands of the gRNA, while the RuvC1 subdomain cleaves the non-complementary strands. Mutations within these subdomains can suppress the nuclease activity of Cas9. For example, mutations D10A and H840A completely inactivate the nuclease activity of Streptococcus pyogenes Cas9 (Jinek et al, Science. 337: 816-821 (2012); Qi et al, Cell. 28; 152 (5): 1173-83 (2013)). As an example, the nuclease-inactivating Cas9 domain comprises the amino acid sequence set forth in the cloning vector pPlatTET-gRNA2 (Accession No. BAV54124).

[0196] The amino acid sequence of an embodiment of catalytically inactivated Cas9 (dCas9) is as follows:

[0197]

[0198] The amino acid sequence of an embodiment of the catalytic Cas9 nickase (nCas9) is as follows:

[0199]

[0200]

[0201] The amino acid sequence of the embodiment of catalytic activation Cas9 is as follows:

[0202]

[0203]

[0204] In some embodiments, Cas9 refers to Cas9 from archaea (e.g., nanoarchaea), which constitute the domain and kingdom of unicellular prokaryotic microorganisms. In some embodiments, the programmable nucleotide binding protein can be a CasX or CasY protein, which has been described in, for example, Burstein et al., "New CRISPR-Cas systems from uncultivated microbes." Cell Res. 2017 Feb 21. doi: 10.1038 / cr.2017.21, the disclosure of which is incorporated by reference in its entirety. Using genome-resolved metagenomics, a variety of CRISPR-Cas systems have been identified, including the first reported Cas9 in the archaeal life domain. This divergent Cas9 protein appears in the little-studied nanoarchaea as part of a CRISPR-Cas system. In bacteria, two previously unknown systems, CRISPR-CasX and CRISPR-CasY, are explored, which are among the most densely compact systems yet to be explored. In some embodiments, in the base editor systems described herein, Cas9 is replaced by CasX or a variant of CasX. In some embodiments, in the base editor systems described herein, Cas9 is replaced by CasY or a variant of CasY. It should be understood that other RNA-guided DNA binding proteins can be used as nucleic acid programmable DNA binding proteins (napDNAbp) and fall within the scope of the disclosure herein.

[0205] In some embodiments, the programmable nucleotide binding protein, also referred to as nucleic acid programmable DNA binding protein (napDNAbp) in the present disclosure, is a CasX protein. In some embodiments, the programmable nucleotide binding protein is a CasY protein. In some embodiments, the programmable nucleotide binding protein comprises an amino acid sequence that is at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or at least 99.5% identical to a native CasX or CasY protein. In some embodiments, the programmable nucleotide binding protein is a native CasX protein or CasY protein. In some embodiments, the programmable nucleotide binding protein comprises an amino acid sequence that is at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or at least 99.5% identical to a CasX protein or CasY protein described herein. It should be understood that according to the disclosure herein, CasX and CasY obtained from other bacterial species can also be used.

[0206] An example of the amino acid sequence of CasX ((uniprot.org / uniprot / F0NN87; uniprot.org / uniprot / F0NH53)tr|F0NN87|F0NN87_SULIHCRISPR-associated CasX protein OS=Sulfolobus islandicus (species is HVE10 / 4) GN=SiH_0402PE=4SV=1) is as follows:

[0207]

[0208] An example of the amino acid sequence of CasX (>tr|F0NH53|F0NH53_SULIR CRISPR-associated protein, Casx OS=Sulfolobus islandicus (species is REY15A) GN=SiRe_0771 PE=4SV=1) is as follows:

[0209]

[0210] Deltaproteobacteria CasX

[0211]

[0212]

[0213] An example of the amino acid sequence of CasY ((ncbi.nlm.nih.gov / protein / APG80656.1)>APG80656.1 CRISPR-associated protein CasY [wild Parcubacteria bacteria]) is as follows:

[0214]

[0215] In some embodiments, the nucleic acid programmable DNA binding protein (napDNAbp) is a single effector of a microbial CRISPR-Cas system. Single effectors of a microbial CRISPR-Cas system include, but are not limited to, Cas9, Cpf1, Cas12b / C2c1, and Cas12c / C2c3. Typically, microbial CRISPR-Cas systems are divided into Class 1 systems and Class 2 systems. Class 1 systems have multi-subunit effector complexes, while Class 2 systems have single protein effectors. For example, Cas9 and Cpf1 are Class 2 systems. In addition to Cas9 and Cpf1, three separate Class 2 CRISPR-Cas systems (Cas12b / C2c1, and Cas12c / C2c3) have been described in Shmakov et al., "Discovery and Functional Characterization of Diverse Class 2 CRISPR Cas Systems," Mol. Cell, 2015 Nov. 5; 60(3): 385-397, the disclosure of which is incorporated herein by reference in its entirety. Two of the effectors in this system, Cas12b / C2c1 and Cas12c / C2c3, contain a Cpf1-associated RuvC-like endonuclease domain. The third system contains an effector with two predicted HEPN RNase domains. The production of mature CRISPR RNA is independent of tracrRNA, unlike the production of CRISPR RNA by Cas12b / C2c1. Cas12b / C2c1 relies on both CRISPR RNA and tracrRNA to perform DNA cleavage.

[0216] The crystal structure of the complex chimeric single-molecule guide RNA (sgRNA) of Alicyclobaccillus acidoterrastris Cas12b / C2c1 (AacC2c1) has been reported. For example, see Liu et al., "C2c1-sgRNA Complex Structure Reveals RNA-Guided DNA Cleavage Mechanism", Mol. Cell, 2017 Jan. 19; 65(2): 310-322, which is incorporated by reference into the disclosure of this specification in its entirety. The crystal structure has also been reported of Alicyclobaccillus acidoterrastris C2c1 bound to the target DNA in a ternary complex. For example, see Yang et al., "PAM-dependent Target DNA Recognition and Cleavage by C2C1 CRISPR-Cas endonuclease", Cell, 2016 Dec. 15; 167(7): 1814-1828, which is incorporated herein by reference in its entirety. The catalytically competent conformation of AacC2c1 with both target and non-target DNA strands is known to be independently located within a single RuvC catalytic pocket, resulting in staggered 7-nucleotide breaks in the target DNA with Cas12b / C2c1-mediated cleavage. Structural comparisons between the Cas12b / C2c1 ternary complex and the previously identified Cas9 and Cpf1 pairs validate the diversity of mechanisms used by the CRISPR-Cas system.

[0217] In some embodiments, the nucleic acid programmable DNA binding protein (napDNAbp) of any fusion protein provided in the present disclosure can be a Cas12b / C2c1 protein, or a Cas12c / C2c3 protein. In some embodiments, napDNAbp is a Cas12b / C2c1 protein. In some embodiments, napDNAbp is a Cas12c / C2c3 protein. In some embodiments, napDNAbp comprises an amino acid sequence that is at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or at least 99.5% identical to a natural Cas12b / C2c1 protein or a Cas12c / C2c3 protein. In some embodiments, napDNAbp is a natural Cas12b / C2c1 protein or a Cas12c / C2c3 protein. In some embodiments, the napDNAbp comprises an amino acid sequence that is at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or at least 99.5% identical to any of the napDNAbp sequences provided in the present disclosure. It should be understood that Cas12b / C2c1 or Cas12c / C2c3 from other species of bacteria may also be used according to the present disclosure.

[0218] An example of the amino acid sequence of Cas12b / C2c1 ((uniprot.org / uniprot / T0D7A2#2) sp|T0D7A2|C2C1_ALIAGCRISPR-associated endonuclease C2c1 OS=Alicyclobacillus acido-terrestris (strain ATCC 49025 / DSM 3922 / CIP 106132 / NCIMB 13137 / GD3B) GN=c2c1 PE=1 SV=1) is as follows:

[0219]

[0220] BhCas12b Bacillus hisashii NCBI reference sequence: WP_095142515

[0221]

[0222] In some embodiments, Cas12b is BvCas12B, which is a variant of BhCas12b and comprises the following changes associated with BhCas12B: S893R, K846R, and E837G.

[0223] BvCas12b (Bacillus sp. V3-13) NCBI reference sequence: WP_101661451.1

[0224]

[0225]

[0226] It should be understood that the polynucleotide programmable nucleotide binding domain may also include a nucleic acid programmable protein that binds RNA. For example, the polynucleotide programmable nucleotide binding domain may be associated with a nucleic acid that guides the polynucleotide programmable nucleotide binding domain to the RNA. Other nucleic acid programmable DNA binding proteins also fall within the scope of this disclosure, but are not specifically listed in this disclosure.

[0227] Cas proteins that can be used in the present disclosure include Class 1 and Class 2. Non-limiting examples of Cas proteins include Cas1, Cas1B, Cas2, Cas3, Cas4, Cas5, Cas5d, Cas5t, Cas5h, Cas5a, Cas6, Cas7, Cas8, Cas9 (also known as Csn1 or Csx12), Cas10, Csy1, Csy2, Csy3, Csy4, Cse1, Cse2, Cse3, Cse4, Cse5e, Csc1, Csc2, Csa5, Csn1, Csn2, Csm1, Csm2, Csm3, Csm4, Csm5, Csm6, Cmr1, Cmr3, Cm Cas12a / Cpfl, Cas12b / C2cl, Cas12c / C2c3, Cas12d / CasY, Cas12e / CasX, Cas12g, Cas12h, and Cas12i, CARF, DinG, their equivalents, or modified versions thereof. Unmodified CRISPR enzymes can have DNA cleavage activity, such as Cas9, which has two functional endonuclease domains: RuvC and HNH. The CRISPR enzyme can direct cleavage of one or both strands of a target sequence, such as within the target sequence and / or within the complement of the target sequence. For example, the CRISPR enzyme can direct cleavage of one or both strands within about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 50, 100, 200, 500, or more base pairs from the first or last nucleotide of the target sequence.

[0228] A vector can be used that encodes a CRISPR enzyme that is mutated relative to the corresponding wild-type enzyme such that the mutated CRISPR enzyme lacks the ability to cleave one or both strands of a target polynucleotide containing a target sequence. Cas9 can refer to a polypeptide having at least or at least about 50%, 60%, 70%, 80%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity and / or sequence homology to a wild-type Cas9 polypeptide embodiment (e.g., Cas9 from Streptococcus pyogenes). Cas9 can refer to a polypeptide having at most or at most about 50%, 60%, 70%, 80%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity and / or sequence homology to a wild-type Cas9 polypeptide embodiment (e.g., Cas9 from Streptococcus pyogenes). Cas9 can refer to a wild-type or modified form of a Cas9 protein, which can include amino acid changes, such as deletions, insertions, substitutions, variations, mutations, fusions, chimeras, or any combination thereof.

[0229] In some embodiments, the methods described herein can utilize genetically engineered Cas proteins. The guide RNA (gRNA) is a short synthetic RNA consisting of the backbone sequence required for Cas binding and a user-defined spacer of about 20 nucleotides that defines the genomic target to be modified. In some embodiments, the backbone comprises GUUUUAGAGC UAGAAAUAGCAAGUUAAAAU AAGGCUAGUC CGUUAUCAAC UUGAAAAAGU GGCACCGAGU CGGUGCUUUU. Whether one skilled in the art can alter the specific genomic target of a Cas protein is determined in part by how specific the gRNA targeting sequence is for the genomic target compared to the rest of the genome.

[0230] The Cas9 nuclease has two functional endonuclease domains: RuvC and HNH. When the target binds, which positions the nuclease domains to cleave opposite strands of the target DNA, Cas9 undergoes a second conformational change. The end result of Cas9-mediated DNA cleavage is a double-strand break (DSB) within the target DNA (approximately 3-4 nucleotides upstream of the PAM sequence). The resulting DSB is then repaired via one of two general repair pathways: (1) the efficient but error-prone nonhomologous end joining (NHEJ) pathway, or (2) the less efficient but highly fidelity homology-directed repair (HDR) pathway.

[0231] The "efficiency" of non-homologous end-joining (NHEJ) and / or homology-directed repair (HDR) can be determined by any convenient method. For example, in some cases, the efficiency can be expressed as a percentage of successful HDR. For example, the inspector nuclease assay can be used to generate cleavage products, and the ratio of product to enzyme substrate can be used to calculate the percentage. For example, the inspector nuclease assay can be used to directly cleave DNA, which is DNA containing newly integrated restriction sequences as a result of successful HDR. More cleaved enzyme substrates indicate a greater percentage HDR (higher efficiency of HDR). As for a specific embodiment, a component (percentage) of HDR can be calculated using the following program [(cleavage product) / (enzyme substrate+cleavage product)] (for example, (b+c) / (a+b+c), where a is the band intensity of the DNA enzyme substrate, and b and c are cleavage products).

[0232] In some cases, efficiency can be expressed as a percentage of successful NHEJ. For example, a T7 endonuclease I assay can be used to generate cleavage products, and the ratio of product to enzyme substrate can be used to calculate percent NHEJ. For example, T7 endonuclease I cleaves mispaired heterodiploid DNA, which is derived from the hybridization of wild-type and mutant DNA strands (NHEJ generates small random insertions or deletions (indels) at the original break site). More cleavages indicate a greater percentage of NHEJ (higher efficiency of NHEJ). As for a specific embodiment, a component (percentage) of NHEJ can be calculated using the following program (1-(1-(b+c) / (a+b+c)) 1 / 2 )×100, where a is the band intensity of the DNA enzyme substrate, and b and c are the cleavage products (Ran et al., Cell. 2013 Sep. 12; 154(6): 1380-9; and Ran et al., Nat Protoc. 2013 Nov.; 8(11): 2281–2308).

[0233] NHEJ repair is the most active repair mechanism, which often results in small nucleotide insertions or deletions (indels) at the DSB site. The stochastic nature of NHEJ-mediated DSB repair has important practical implications, as a population of cells expressing Cas9 and gRNA or guide polynucleotides may result in a diverse set of mutations. In most cases, NHEJ produces small insertions or deletions in the target DNA, resulting in amino acid deletions, insertions, or frameshift mutations, leading to the generation of premature stop codons within the open reading frame (ORF) of the targeted gene. The ideal end result is a loss-of-function mutation within the targeted gene.

[0234] Although NHEJ-mediated DSB repair often disrupts the open reading frame of a gene, homology-directed repair (HDR), similar to the addition of fluorophores or tags, can be used to generate specific nucleotide changes ranging from single nucleotide changes to large insertions.

[0235] To use HDR for gene editing, a DNA repair template containing the desired sequence can be delivered into the cell type of interest using gRNA and Cas9 or Cas9 nickase. The repair template can contain the desired edit, as well as additional homologous sequences (named left and right homology arms) immediately upstream and downstream of the target. The length of each homology arm can depend on the size of the introduced variation, with larger insertions requiring longer homology arms. The repair template can be a single-stranded oligonucleotide, a double-stranded oligonucleotide, or a double-stranded DNA plasmid. The efficiency of HDR is typically low (less than 10% of the modified allele), even for cells expressing Cas9, gRNA, and exogenous repair templates. The efficiency of HDR can be improved by synchronizing the cells because HDR occurs during the S and G2 phases of the cell cycle. Chemically or genetically inhibiting genes involved in NHEJ also improves HDR efficiency.

[0236] In some embodiments, Cas9 is a modified Cas9. Throughout the genome (where partial homology exists), there may be additional sites for a given gRNA targeting sequence. These sites are referred to as off-targets and must be considered when designing gRNAs. In addition to optimizing gRNA design, CRISPR specificity can also be improved through modifications to Cas9. Cas9 generates double-strand breaks (DSBs) through the combined activity of two nuclease domains, RuvC and HNH. Cas9 nickase, i.e., the D10A mutant of SpCas9, retains a nuclease domain that generates DNA nicks instead of DSBs. Nickase systems can also be combined with HDR-mediated gene editing for specific gene editing.

[0237] In some embodiments, the modified Cas9 is a high-fidelity Cas9 enzyme. In some embodiments, the high-fidelity Cas9 enzyme is SpCas9 (K855A), eSpCas9 (1.1), SpCas9-HF1, or a super-accurate Cas9 variant (HypaCas9). The modified Cas9 eSpCas9 (1.1) contains an alanine substitution that weakens the interaction between the HNH / RuvC groove and the non-target DNA strand, preventing the separation of the strands and cutting at off-target sites. Similarly, SpCas9-HF1 reduces off-target editing by alanine substitution, which disrupts the interaction of Cas9 with the DNA phosphate backbone. HypaCas9 contains mutations in the REC3 domain (SpCas9 N692A / M694A / Q695A / H698A), which increase Cas9 correction and target discrimination. All three high-fidelity enzymes generate fewer off-target edits than wild-type Cas9. The amino acid sequences of high-fidelity Cas9 embodiments are provided below. In this sequence, high-fidelity Cas9 domain mutations relative to the reference Cas9 are shown in bold and underlined:

[0238]

[0239]

[0240] In some cases, Cas9 is a variant Cas9 protein. When compared to the amino acid sequence of the wild-type Cas9 protein, the variant Cas9 polypeptide has an amino acid sequence that differs by one amino acid (e.g., having a deletion, insertion, substitution, fusion). In some cases, the variant Cas9 polypeptide has an amino acid change (e.g., a deletion, insertion, or substitution) that reduces the nuclease activity of the Cas9 polypeptide. For example, in some cases, the variant Cas9 polypeptide has less than 50%, less than 40%, less than 30%, less than 20%, less than 10%, less than 5%, or less than 1% of the nuclease activity of the corresponding wild-type Cas9 protein. In some cases, the variant Cas9 protein has substantially no nuclease activity. When the subject Cas9 protein is a variant Cas9 protein that has substantially no nuclease activity, it can be referred to as "dCas9".

[0241] In some cases, the variant Cas9 protein has reduced nuclease activity. For example, the variant Cas9 protein has less than about 20%, less than about 15%, less than about 10%, less than about 5%, less than about 1%, less than about %, or less than about 0.1% of the endonuclease activity of the corresponding wild-type Cas9 protein.

[0242] In some cases, the variant Cas9 protein can cleave the complementary strand of the guide target sequence, but has a reduced ability to cleave the non-complementary strand of the double-stranded guide target sequence. For example, the variant Cas9 protein may have a mutation (amino acid substitution) that reduces the function of the RuvC domain. As a non-limiting example, in some embodiments, the variant Cas9 protein has D10A (aspartic acid at amino acid position 10 is changed to alanine), thereby being able to cleave the complementary strand of the double-stranded guide target sequence, but having a reduced ability to cleave the non-complementary strand of the double-stranded guide target sequence (so that when the variant Cas9 protein cleaves the double-stranded target nucleic acid, the result is a single strand break (SSB) rather than a double strand break (DSB)) (for example, refer to Jinek et al., Science. 2012 Aug. 17; 337(6096): 816-21).

[0243] In some cases, the variant Cas9 protein can cleave the non-complementary strand of the guide target sequence, but has a reduced ability to cleave the complementary strand of the double-stranded guide target sequence. For example, the variant Cas9 protein may have a mutation (amino acid substitution) that reduces the function of the HNH domain (RuvC / HNH / RuvC domain motif). As a non-limiting example, in some embodiments, the variant Cas9 protein has an H840A (histidine at amino acid position 840 becomes alanine) mutation, thereby being able to cleave the non-complementary strand of the guide target sequence, but has a reduced ability to cleave the complementary strand of the double-stranded guide target sequence (such that when the variant Cas9 protein cleaves the double-stranded target nucleic acid, the result is a single strand break (SSB) rather than a double strand break (DSB)). Such a Cas9 protein has a reduced ability to cleave a guide target sequence (e.g., a single guide target sequence), but retains the ability to bind to a guide target sequence (e.g., a single guide target sequence).

[0244] In some cases, the variant Cas9 protein has a reduced ability to cleave both the complementary and non-complementary strands of a double-stranded target DNA. As a non-limiting example, in some embodiments, the variant Cas9 protein has both the D10A and H840A mutations, so that the polypeptide has a reduced ability to cleave both the complementary and non-complementary strands of a double-stranded target DNA. Such a Cas9 protein has a reduced ability to cleave a target DNA (e.g., a single-stranded target DNA), but retains the ability to bind to a target DNA (e.g., a single-stranded target DNA).

[0245] As another non-limiting example, in some cases, the variant Cas9 protein has W476A and W1126A mutations, so that the polypeptide has a reduced ability to cleave target DNA. Such a Cas9 protein has a reduced ability to cleave target DNA (e.g., single-stranded target DNA), but retains the ability to bind to target DNA (e.g., single-stranded target DNA).

[0246] As another non-limiting example, in some cases, the variant Cas9 protein has P475A, W476A, N477A, ​​D1125A, W1126A, and D1127A mutations, so that the polypeptide has a reduced ability to cleave target DNA. Such Cas9 protein has a reduced ability to cleave target DNA (e.g., single-stranded target DNA), but retains the ability to bind to target DNA (e.g., single-stranded target DNA).

[0247] As for another non-limiting embodiment, in some cases, the variant Cas9 protein has H840A, W476A and W1126A mutations, whereby the polypeptide has a reduced ability to cleave target DNA. Such Cas9 proteins have a reduced ability to cleave target DNA (e.g., single-stranded target DNA), but retain the ability to bind to target DNA (e.g., single-stranded target DNA). As for another non-limiting embodiment, in some cases, the variant Cas9 protein has H840A, D10A, W476A and W1126A mutations, whereby the polypeptide has a reduced ability to cleave target DNA. Such Cas9 proteins have a reduced ability to cleave target DNA (e.g., single-stranded target DNA), but retain the ability to bind to target DNA (e.g., single-stranded target DNA). In some embodiments, the variant Cas9 has a repaired catalytic His residue at position 840 (A840H) of the Cas9 HNH domain.

[0248] As for another non-limiting embodiment, in some cases, the variant Cas9 protein has H840A, P475A, W476A, N477A, ​​D1125A, W1126A, and D1127A mutations, so that the polypeptide has a reduced ability to cleave target DNA. Such Cas9 proteins have a reduced ability to cleave target DNA (e.g., single-stranded target DNA), but retain the ability to bind to target DNA (e.g., single-stranded target DNA). As for another non-limiting embodiment, in some cases, the variant Cas9 protein has D10A, H840A, P475A, W476A, N477A, ​​D1125A, W1126A, and D1127A mutations, so that the polypeptide has a reduced ability to cleave target DNA. Such Cas9 proteins have a reduced ability to cleave target DNA (e.g., single-stranded target DNA), but retain the ability to bind to target DNA (e.g., single-stranded target DNA). In some cases, when the variant Cas9 protein has W476A and W1126A mutations, or when the variant Cas9 protein has P475A, W476A, N477A, ​​D1125A, W1126A, and D1127A mutations, the variant Cas9 protein cannot effectively bind to the PAM sequence. Therefore, in some cases, when such a variant Cas9 protein is used in a binding method, the method does not require a PAM sequence. In other words, in some cases, when such a variant Cas9 protein is used in a binding method, the method may include a guide RNA (gRNA), but the method can be performed in the absence of a PAM sequence (and therefore, the binding specificity is provided by the target segment of the guide RNA). Other residues can be mutated to achieve the above effects (i.e., inactivate one or another nuclease portion). As a non-limiting example, residues D10, G12, G17, E762, H840, N854, N863, H982, H983, A984, D986, and / or A987 may be altered (i.e., substituted). In addition, mutations other than alanine substitutions are also suitable.

[0249] In some embodiments, a variant Cas9 protein with reduced catalytic activity (e.g., when the Cas9 protein has D10, G12, G17, E762, H840, N854, N863, H982, H983, A984, D986, and / or A987 mutations, e.g., D10A, G12A, G17A, E762A, H840A, N854A, N863A, H982A, H983A, A984A, and / or D986A), the variant Cas9 protein can still bind to the target DNA in a site-specific manner (because it is still guided to the target DNA sequence by the guide RNA), as long as it still retains the ability to interact with the guide RNA.

[0250] As an alternative to S. pyogenes, Cas9 may include an RNA-guided endonuclease from the Cpf1 family, which exhibits cleavage activity in mammalian cells. CRISPR from Prevotella and Francisella 1 (CRISPR / Cpf1) is a DNA editing technology similar to the CRISPR / Cas system. Cpf1 is an RNA-guided endonuclease of the class II CRISPR / Cas system. This acquired immune mechanism is present in Prevotella and Francisella. The Cpf1 gene is associated with the CRISPR locus and encodes an endonuclease that uses a guide RNA to find and cleave viral DNA. Cpf1 is a smaller and simpler endonuclease than Cas9, overcoming several limitations of the CRISPR / Cas system. Unlike the Cas9 nuclease, Cpf1 mediates DNA cleavage as double-strand breaks with short 3' overhangs. The staggered cleavage pattern of Cpf1 can open up the possibility of targeted gene transfer (similar to traditional restriction enzyme cloning), which can improve gene editing efficiency. Similar to the aforementioned Cas9 variants and homologs, Cpf1 can also expand the number of sites targeted by CRISPR to AT-rich regions or AT-rich genomes (which lack the NGGPAM site preferred by SpCas9). The Cpf1 locus contains a fixed α / β domain, RuvC-I, followed by a helical region, RuvC-II, and a zinc finger domain. The Cpf1 protein has a RuvC-like nuclease domain, which is similar to the RuvC domain of Cas9. Furthermore, Cpf1 does not have a HNH nuclease domain, and the N-terminus of Cpf1 does not have the α-helical recognition lobe of Cas9. The Cpf1 CRISPR-Cas domain architecture shows that Cpf1 is functionally unique and is classified as a Class 2, Type V CRISPR system. The Cpf1 locus encodes Cas1, Cas2, and Cas4 proteins that are more similar to type I and III than to type II systems. Functional Cpf1 does not require transactivating CRISPR RNA (tracrRNA), so only CRISPR (crRNA) is required. This facilitates genome editing because Cpf1 is not only smaller than Cas9, but also has a smaller sgRNA molecule (about half the number of nucleotides as Cas9). The Cpf1-crRNA complex cleaves the target DNA or RNA by recognizing the protospacer-adjacent motif 5'-YTN-3' (as opposed to the G-rich PAM targeted by Cas9). After recognizing the PAM, Cpf1 introduces sticky-end DNA double-strand breaks with 4 or 5 nucleotide overhangs.

[0251] Certain aspects of the present disclosure provide fusion proteins comprising a domain that is a nucleic acid programmable DNA binding protein that can be used to guide proteins (such as base editors) to specific nucleic acid (e.g., DNA or RNA) sequences. In specific embodiments, the fusion protein comprises a nucleic acid programmable DNA binding protein domain and a deaminase domain. DNA binding proteins include, but are not limited to, Cas9 (e.g., dCas9 and nCas9), Cas12a / Cpfl, Cas12b / C2cl, Cas12c / C2c3, Cas12d / CasY, Cas12e / CasX, Cas12g, Cas12h, and Cas12i. An example of a nucleic acid programmable DNA binding protein with a different PAM specificity from Cas9 is a clustered regularly interspaced short palindromic repeat (CRISPR) from Prevotella and Francisella 1 (Cpf1). Similar to Cas9, Cpf1 is also a Class 2 CRISPR effector. Cpf1 has been shown to mediate robust DNA interference with different properties from Cas9. Cpf1 is a single RNA-guided endonuclease lacking tracrRNA that utilizes a T-rich protospacer adjacent motif (TTN, TTTN, or YTN). In addition, Cpf1 cleaves DNA by staggered DNA double-strand breaks. Of the 16 Cpf1 family proteins, two enzymes from Acidaminococcus and Lachnospiraceae were shown to have potent genome editing activity in human cells. Cpf1 proteins are known in the industry and have been previously described, for example, Yamano et al., "Crystal structure of Cpf1 in complex with guide RNA and target DNA." Cell (165) 2016, p. 949-962; the entire disclosure of this specification is incorporated by reference.

[0252] Also useful in the present compositions and methods are nuclease-inactive Cpf1 (dCpf1) variants, which can be used as guide nucleotide sequence programmable DNA binding protein domains. The Cpf1 protein has a RuvC-like nuclease domain, which is similar to the RuvC domain of Cas9, but does not have an HNH nuclease domain, and the N-terminus of Cpf1 does not have the α-helical recognition lobe of Cas9. It is shown in Zetsche et al., Cell, 163, 759-771, 2015 (the disclosure of which is incorporated by reference into this specification) that the RuvC-like domain of Cpf1 is responsible for splitting the two strands of DNA, and the inactivation of the RuvC-like domain inactivates the nuclease activity of Cpf1. For example, mutations corresponding to D917A, E1006A, or D1255A in Francisella novicida Cpf1 inactivate the nuclease activity of Cpf1. In some embodiments, the disclosed dCpf1 comprises a mutation corresponding to D917A, E1006A, D1255A, D917A / E1006A, D917A / D1255A, E1006A / D1255A, or D917A / E1006A / D1255A. It should be understood that any mutation that inactivates the RuvC domain of Cpf1, such as a substitution mutation, a deletion, or an insertion, can be used according to the disclosure herein.

[0253] In some embodiments, the nucleic acid programmable DNA binding protein (napDNAbp) of any fusion protein provided in the present disclosure can be a Cpf1 protein. In some embodiments, the Cpf1 protein is a Cpf1 nickase (nCpf1). In some embodiments, the Cpf1 protein is a nuclease-inactivated Cpf1 (dCpf1). In some embodiments, Cpf1, nCpf1, or dCpf1 comprises an amino acid sequence that is at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or at least 99.5% identical to the Cpf1 sequence disclosed herein. In some embodiments, dCpf1 comprises an amino acid sequence that is at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or at least 99.5% identical to the Cpf1 sequence disclosed herein, and comprises mutations corresponding to D917A, E1006A, D1255A, D917A / E1006A, D917A / D1255A, E1006A / D1255A, or D917A / E1006A / D1255A. It should be understood that Cpf1 from other species may also be used according to the disclosure herein.

[0254] The amino acid sequence of wild-type Francisella novo Cpf1 is as follows: D917, E1006, and D1255 are in bold and underlined.

[0255]

[0256] The amino acid sequence of Francisella novo Cpf1 D917A is as follows (A917, E1006, and D1255 are in bold and underlined).

[0257]

[0258] The amino acid sequence of Francisella novo Cpf1 E1006A is as follows (D917, A1006, and D1255 are in bold and underlined).

[0259]

[0260] The amino acid sequence of Francisella novo Cpf1 D1255A is as follows (the positions of the D917, E1006, and A1255 mutations are in bold and underlined).

[0261]

[0262] The amino acid sequence of Francisella novo Cpf1 D917A / E1006A is as follows (A917, A1006, and D1255 are in bold and underlined).

[0263]

[0264]

[0265] The amino acid sequence of Francisella novo Cpf1 D917A / D1255A is as follows (A917, E1006, and A1255 are in bold and underlined).

[0266]

[0267] The amino acid sequence of Francisella novo Cpf1 E1006A / D1255A is as follows (D917, A1006, and A1255 are in bold and underlined).

[0268]

[0269] The amino acid sequence of Francisella novo Cpf1 D917A / E1006A / D1255A is as follows (A917, A1006, and A1255 are in bold and underlined).

[0270]

[0271]

[0272] In some embodiments, the variant Cas protein can be spCas9, spCas9-VRQR, spCas9-VRER, xCas9(sp), saCas9, saCas9-KKH, spCas9-MQKSER, spCas9-LRKIQK, or spCas9-LRVSQL.

[0273] The amino acid sequence of an embodiment of SaCas9 is as follows:

[0274]

[0275]

[0276] In this sequence, the underlined and bold residue N579 can be mutated (e.g., to A579) to obtain a SaCas9 nickase.

[0277] The amino acid sequence of an embodiment of SaCas9n is as follows:

[0278]

[0279] In this sequence, residue A579 (which can be mutated from N579 to obtain the SaCas9 nickase) is underlined and in bold. The amino acid sequence of an example of SaKKH Cas9 is as follows:

[0280]

[0281]

[0282] Residue A579 (which can be mutated from N579 to obtain the SaCas9 nickase) is underlined and in bold as above. Residues K781, K967, and H1014 (which can be mutated from E781, N967, and R1014 to obtain SaKKH Cas9) are underlined and in italics as above.

[0283] The polynucleotide programmable nucleotide binding domain of the base editor may itself include one or more domains. For example, the polynucleotide programmable nucleotide binding domain may itself include one or more nuclease domains. In some embodiments, the nuclease domain of the polynucleotide programmable nucleotide binding domain may include an endonuclease or an exonuclease. In the present disclosure, the term "exonuclease" refers to a protein or polypeptide that can digest a nucleic acid (e.g., RNA or DNA) from a free end, and the term "endonuclease" refers to a protein or polypeptide that can catalyze (e.g., split) an inner region of a nucleic acid (e.g., DNA or RNA). In some embodiments, an endonuclease can cleave a single strand of a double-stranded nucleic acid. In some embodiments, an endonuclease can cleave two strands of a double-stranded nucleic acid molecule. In some embodiments, the polynucleotide programmable nucleotide binding domain may be a deoxyribonuclease. In some embodiments, the polynucleotide programmable nucleotide binding domain may be a ribonuclease.

[0284] In some embodiments, the nuclease domain of the polynucleotide programmable nucleotide binding domain can cut zero, one, or two strands of the target nucleotide. In some cases, the polynucleotide programmable nucleotide binding domain may include a nickase domain. In the present disclosure, the term "nickase" refers to a polynucleotide programmable nucleotide binding domain comprising a nuclease domain that can only split one of the two strands in a diploid nucleic acid molecule (e.g., DNA). In some embodiments, the nickase can be derived from a fully catalytically active (e.g., natural) form of the polynucleotide programmable nucleotide binding domain by introducing one or more mutations into the active polynucleotide programmable nucleotide binding domain. For example, when the polynucleotide programmable nucleotide binding domain comprises a nickase domain derived from Cas9, the Cas9-derived nickase domain may include a D10A mutation and a histidine at position 840. In this case, residue H840 retains catalytic activity and can thus cleave a single strand of a nucleic acid duplex. In another embodiment, the Cas9-derived nickase domain may include an H840A mutation, while the amino acid residue at position 10 remains D. In some embodiments, a nickase may be derived from a fully catalytically active (e.g., native) form of a polynucleotide programmable nucleotide binding domain by removing all or part of a nuclease domain that is not required for nickase activity. For example, when the polynucleotide programmable nucleotide binding domain comprises a nickase domain derived from Cas9, the Cas9-derived nickase domain may comprise a deletion of all or part of a RuvC domain or a HNH domain.

[0285] Thus, a base editor comprising a polynucleotide programmable nucleotide binding domain including a nickase domain can generate a single-stranded DNA break (nick) at a specific polynucleotide target sequence (e.g., as determined by the complementary sequence of the bound guide nucleic acid). In some embodiments, the strand of the nucleic acid diploid target polynucleotide sequence cleaved by a base editor comprising a nickase domain (e.g., a Cas9-derived nickase domain) is the strand that is not edited by the base editor (i.e., the strand cleaved by the base editor is opposite to the strand containing the base to be edited). In other embodiments, a base editor comprising a nickase domain (e.g., a Cas9-derived nickase domain) can cleave the strand of a DNA molecule targeted for editing. In this case, the untargeted strand will not be cleaved.

[0286] The present disclosure also provides a base editor comprising a polynucleotide programmable nucleotide binding domain that is catalytically dead (i.e., unable to cleave a target polynucleotide sequence). In the present disclosure, the terms "catalytically dead" and "nuclease dead" are used interchangeably to refer to a polynucleotide programmable nucleotide binding domain that has one or more mutations and / or deletions, resulting in its inability to cleave a strand of nucleic acid. In some embodiments, the catalytically dead polynucleotide programmable nucleotide binding domain base editor may lack nuclease activity as a result of specific point mutations in one or more nuclease domains. For example, taking a base editor comprising a Cas9 domain as an example, Cas9 may comprise both a D10A mutation and an H840A mutation. These mutations inactivate both nuclease domains, thereby resulting in a loss of nuclease activity. In other embodiments, the catalytically dead polynucleotide programmable nucleotide binding domain may comprise one or more deletions of all or part of a catalytic domain (e.g., RuvC1 and / or HNH domain). In further embodiments, the catalytically dead polynucleotide programmable nucleotide binding domain comprises a point mutation (eg, D10A or H840A), and a deletion of all or part of the nuclease domain.

[0287] It is expected that mutations that can generate catalytically dead polynucleotide programmable nucleotide binding domains from the polynucleotide programmable nucleotide binding domains of the previous functional version are also covered in this disclosure. For example, taking catalytically dead Cas9 (dCas9) as an example, variants with mutations other than D10A and H840A are provided, which result in nuclease inactivation Cas9. For example, these mutations include other amino acid substitutions in D10A and H840A, or other substitutions in the nuclease domain of Cas9 (e.g., substitutions in the HNH nuclease subdomain and / or the RuvC1 subdomain). Based on the disclosure herein and field knowledge, additional appropriate nuclease inactivation dCas9 domains are obvious to those skilled in the art and fall within the scope disclosed herein. Additional embodiments of these appropriate nuclease inactivation Cas9 domains include, but are not limited to, D10A / H840A, D10A / D839A / H840A, and D10A / D839A / H840A / N863A mutation domains. (For example, see Prashant et al., "CAS9 transcriptional activators for target specificity screening and paired nickases for cooperative genome engineering". Nature Biotechnology. 2013; 31(9): 833-838, which is incorporated herein by reference in its entirety.) In some embodiments, the dCas9 domain comprises an amino acid sequence that is at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or at least 99.5% identical to any of the dCas9 domains provided in the present disclosure. In some embodiments, the dCas9 domain comprises an amino acid sequence having 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 21, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50 or more mutations compared to any of the amino acid sequences listed in the disclosure.In some embodiments, the dCas9 domain comprises an amino acid sequence having at least 10, at least 15, at least 20, at least 30, at least 40, at least 50, at least 60, at least 70, at least 80, at least 90, at least 100, at least 150, at least 200, at least 250, at least 300, at least 350, at least 400, at least 500, at least 600, at least 700, at least 800, at least 900, at least 1000, at least 1100, or at least 1200 identical contiguous amino acid residues compared to any of the amino acid sequences listed in the disclosure.

[0288] Non-limiting examples of polynucleotide programmable nucleotide binding domains that can be incorporated into base editors include CRISPR protein-derived domains, cleavage nucleases, meganucleases, TAL nucleases (TALENs), and zinc finger nucleases (ZFNs). In some cases, a base editor comprises a polynucleotide programmable nucleotide binding domain comprising a natural or modified protein or portion thereof that, through a bound guide nucleic acid, is capable of binding to a nucleic acid sequence during CRISPR (i.e., clustered regularly interspaced short palindromic repeats)-mediated nucleic acid modification. Such a protein is referred to as a "CRISPR protein." Therefore, disclosed herein is a base editor comprising a polynucleotide programmable nucleotide binding domain comprising all or part of a CRISPR protein (i.e., a base editor comprising all or part of a CRISPR protein as a domain, also referred to as a "CRISPR protein-derived domain" of a base editor). The CRISPR protein-derived domain incorporated into a base editor can be modified compared to a wild-type or natural version of a CRISPR protein. For example, as described below, a CRISPR protein-derived domain may comprise one or more mutations, insertions, deletions, rearrangements, and / or recombination compared to a wild-type or naturally occurring version of a CRISPR protein.

[0289] In some embodiments, the CRISPR protein-derived domain incorporated into the base editor is an endonuclease (e.g., a deoxyribonuclease or a ribonuclease) that is capable of binding to a target polynucleotide when combined with a bound guide nucleic acid. In some embodiments, the CRISPR protein-derived domain incorporated into the base editor is a nickase that is capable of binding to a target polynucleotide when combined with a bound guide nucleic acid. In some embodiments, the CRISPR protein-derived domain incorporated into the base editor is a catalytic death domain that is capable of binding to a target polynucleotide when combined with a bound guide nucleic acid. In some embodiments, the target polynucleotide bound by the CRISPR protein-derived domain of the base editor is DNA. In some embodiments, the target polynucleotide bound by the CRISPR protein-derived domain of the base editor is RNA.

[0290] In some embodiments, the CRISPR protein-derived domain of the base editor may include all or part of Cas9 obtained from the following: Corynebacterium ulcerans (NCBI Refs: NC_015683.1, NC_017317.1); Corynebacterium diphtheriae (NCBI Refs: NC_016782.1, NC_016786.1); Spiroplasma aphidophila (NCBI Ref: NC_021284.1); Prevotella intermedia (NCBI Ref: NC_017861.1); Spiroplasma punctatum (NCBI Ref: NC_021846.1); Streptococcus iniae (NCBI Ref: NC_021314.1); Bellella balticola (NCBI Ref: NC_018010.1); Campylobacter thermophilus (NCBI Ref: NC_018721.1); Streptococcus thermophilus (NCBI Ref: NC_021846.1); Streptococcus iniae (NCBI Ref: NC_021314.1); Ref: YP_820832.1); Listeria innocua (NCBI Ref: NP_472073.1); Campylobacter jejuni (NCBI Ref: YP_002344900.1); Neisseria meningitidis (NCBI Ref: YP_002342100.1); Streptococcus pyogenes; or Staphylococcus aureus.

[0291] In some embodiments, the Cas9 domain is a Cas9 domain from Staphylococcus aureus (SaCas9). In some embodiments, SaCas9 is a nuclease active SaCas9, a nuclease inactive SaCas9 (SaCas9d), or a SaCas9 nickase (SaCas9n). In some embodiments, SaCas9 comprises an N579A mutation, or a corresponding mutation in any of the amino acid sequences provided in the present disclosure.

[0292] In some embodiments, the SaCas9 domain, SaCas9d domain, or SaCas9n domain can bind to a nucleic acid sequence having a non-canonical PAM. In some embodiments, the SaCas9 domain, SaCas9d domain, or SaCas9n domain can bind to a nucleic acid sequence having a NNGRRT or NNNRRT PAM sequence. In some embodiments, the SaCas9 domain comprises one or more of E781X, N967X, and R1014X mutations, or corresponding mutations in any amino acid sequence provided in the present disclosure, wherein X is any amino acid. In some embodiments, the SaCas9 domain comprises one or more of E781K, N967K, and R1014H mutations, or one or more corresponding mutations in any amino acid sequence provided in the present disclosure. In some embodiments, the SaCas9 domain comprises E781K, N967K, and R1014H mutations, or corresponding mutations in any amino acid sequence provided in the present disclosure.

[0293] The base editor may comprise a domain derived from all or part of a Cas9 that is a high-fidelity Cas9. In some embodiments, the high-fidelity Cas9 of the base editor is an engineered Cas9 domain comprising one or more mutations that reduce the electrostatic interaction between the Cas9 domain and the sugar-phosphate backbone of DNA compared to the corresponding wild-type Cas9 domain. A Cas9 domain with reduced electrostatic interactions with the sugar-phosphate backbone of DNA may have fewer off-target effects. In some embodiments, a Cas9 domain (e.g., a wild-type Cas9 domain) comprises one or more mutations that reduce the association between the Cas9 domain and the sugar-phosphate backbone of DNA. In some embodiments, the Cas9 domain comprises one or more mutations that reduce the association of the Cas9 domain with the sugar-phosphate backbone of DNA by at least 1%, at least 2%, at least 3%, at least 4%, at least 5%, at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, or more.

[0294] As used in this disclosure, the term "guide polynucleotide" refers to a polynucleotide that is specific for a target sequence and can form a complex with a polynucleotide programmable nucleotide binding domain protein (e.g., Cas9 or Cpf1). In one embodiment, the guide polynucleotide is a guide RNA. As used in this disclosure, the term "guide RNA (gRNA)" and its grammatical synonyms may refer to an RNA that is specific for a target DNA and can form a complex with a Cas protein. The RNA / Cas complex can assist in "guiding" the Cas protein to the target DNA. Cas9 / crRNA / tracrRNA endonucleolytically cleaves a linear or circular dsDNA target that is complementary to the spacer. Target strands that are not complementary to the crRNA are first cleaved endonucleolytically and then 3'-5' trimmed exonucleolytically. In nature, DNA binding and cleavage typically requires proteins and two RNAs. However, a single guide RNA ("sgRNA" or simply "gRNA") can be genetically engineered to combine aspects of both crRNA and tracrRNA into a single RNA. For example, see Jinek M., Chylinski K., Fonfara I., Hauer M., Doudna JA, Charpentier E. Science 337:816-821 (2012), which is incorporated herein by reference in its entirety. Cas9 recognizes short motifs (PAM or protospacer adjacent motifs) in CRISPR repeat sequences to help distinguish self from non-self.

[0295] In some embodiments, the guide polynucleotide is at least one single guide RNA ("sgRNA" or "gRNA"). In some embodiments, the guide polynucleotide is at least one tracrRNA. In some embodiments, the guide polynucleotide does not require a PAM sequence to guide the polynucleotide programmable DNA binding domain (e.g., Cas9 or Cpf1) to the target nucleotide sequence.

[0296] The polynucleotide programmable nucleotide binding domain (e.g., CRISPR-derived domain) of the base editor disclosed herein can recognize a target nucleotide sequence by associating with a guide polynucleotide. The guide polynucleotide (e.g., gRNA) is typically single-stranded and can be programmed to site-specifically bind (i.e., by complementary base pairing) to a target sequence of a polynucleotide, thereby guiding the base editor (which is together with the guide nucleic acid) to the target sequence. The guide polynucleotide can be DNA. The guide polynucleotide can be RNA. In some cases, the guide polynucleotide comprises a natural nucleotide (e.g., adenosine). In some cases, the guide polynucleotide comprises a non-natural (or non-natural) nucleotide (e.g., a peptide nucleic acid or a nucleotide analog). In some cases, the target region length of the guide nucleic acid sequence can be at least 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30 nucleotides. The target region length of the guide nucleic acid can be 10 to 30 nucleotides long, or 15 to 25 nucleotides long, or 15 to 20 nucleotides long.

[0297] In some embodiments, the guide polynucleotide comprises two or more individual polynucleotides that can interact with each other through, for example, complementary base pairing (e.g., dual guide polynucleotides). For example, the guide polynucleotide can comprise a CRISPR RNA (crRNA) and a trans-activating CRISPR RNA (tracrRNA). For example, the guide polynucleotide can comprise one or more trans-activating CRISPR RNAs (tracrRNAs).

[0298] In a type II CRISPR system, targeting of nucleic acids by a CRISPR protein (e.g., Cas9) typically requires complementary base pairing between a first RNA molecule (crRNA) comprising a sequence that recognizes the target sequence and a second RNA molecule (trRNA) comprising a repeat sequence that forms a backbone region that stabilizes the guide RNA-CRISPR protein complex. Such a dual guide RNA system can be employed as a guide polynucleotide to direct the base editors disclosed herein to the target nucleotide sequence.

[0299] In some embodiments, the base editors provided in the present disclosure utilize a single guide polynucleotide (e.g., gRNA). In some embodiments, the base editors provided in the present disclosure utilize dual guide polynucleotides (e.g., dual gRNA). In some embodiments, the base editors provided in the present disclosure utilize one or more guide polynucleotides (e.g., a plurality of gRNAs). In some embodiments, a single guide polynucleotide is applied to different base editors described herein. For example, a single guide polynucleotide can be applied to a cytidine base editor and an adenosine base editor.

[0300] In other embodiments, the guide polynucleotide may include both the polynucleotide target portion of the nucleic acid and the backbone portion of the nucleic acid in a single molecule (i.e., a single-molecule guide nucleic acid). For example, the single-molecule guide polynucleotide can be a single guide RNA (sgRNA or gRNA). In the present disclosure, the term guide polynucleotide sequence is intended to cover any single-, double-, or multi-molecule nucleic acid that can interact with a base editor and guide the base editor to a target polynucleotide sequence.

[0301] Typically, a guide polynucleotide (e.g., a crRNA / trRNA complex or a gRNA) comprises a "polynucleotide target segment" that includes a sequence capable of recognizing and binding to a target polynucleotide sequence, and a "protein binding segment" that stabilizes the polynucleotide programmable nucleotide binding domain component of the base editor within the guide polynucleotide. In some embodiments, the polynucleotide target segment of the guide polynucleotide recognizes and binds to a DNA polynucleotide, thereby facilitating base editing in the DNA. In other cases, the polynucleotide target segment of the guide polynucleotide recognizes and binds to an RNA polynucleotide, thereby facilitating base editing in the RNA. In the present disclosure, a "segment" refers to a section or region of a molecule, for example, a contiguous stretch of nucleotides in a guide polynucleotide. A segment also refers to a region / segment of a complex, such that a segment may include a region of more than one molecule. For example, when the guide polynucleotide comprises a plurality of nucleic acid molecules, the protein binding segment may include all or part of a plurality of separate molecules, which hybridize, for example, along a complementary region. In some embodiments, a protein-binding segment of a DNA target RNA comprising two separate molecules may include (i) base pairs 40-75 of a first RNA molecule of 100 base pairs in length; and (ii) base pairs 10-25 of a second RNA molecule of 50 base pairs in length. Unless otherwise specifically defined in a particular context, the definition of "segment" is not limited to a specific number of total base pairs, is not limited to any specific number of base pairs from a given RNA molecule, is not limited to a specific number of separate molecules within a complex, and may include regions of RNA molecules having any total length, and may include regions having complementarity with other molecules.

[0302] The guide RNA or guide polynucleotide may comprise two or more RNAs, for example, CRISPR RNA (crRNA) and trans-activating CRISPR RNA (tracrRNA). The guide RNA or guide polynucleotide may occasionally comprise single-stranded RNA, or a single guide RNA (sgRNA) generated by fusion of a portion (e.g., a functional portion) of crRNA and tracrRNA. The guide RNA or guide polynucleotide may also be a dual RNA of crRNA and tracrRNA. In addition, crRNA may hybridize with target DNA.

[0303] As discussed above, the guide RNA or guide polynucleotide can be an expression product. For example, the DNA encoding the guide RNA can be a vector comprising a sequence encoding the guide RNA. The guide RNA or guide polynucleotide can be transferred into the cell by transfecting the cell with an isolated guide RNA or a plastid DNA comprising a sequence encoding the guide RNA, and a promoter. The guide RNA or guide polynucleotide can also be transferred into the cell in other ways, such as using viral-mediated gene delivery.

[0304] The guide RNA or guide polynucleotide can be isolated. For example, the guide RNA can be transferred into a cell or organism in the form of isolated RNA. The guide RNA can be made by in vitro transcription using any in vitro transcription system known in the art. The guide RNA can be transferred to the cell in the form of isolated RNA, rather than in the form of a plastid containing the coding sequence.

[0305] The guide RNA or guide polynucleotide may comprise three regions: a first region at the 5' end, which may be complementary to a target site in a chromosomal sequence; a second internal region, which may form a stem-loop structure; and a third 3' region, which may be single-stranded. The first region of each guide RNA may also be different, so that each guide RNA guides the fusion protein to a specific target site. In addition, the second and third regions of each guide RNA may be the same in all guide RNAs.

[0306] The first region of the guide RNA or guide polynucleotide may be complementary to the sequence of the target site in the chromosome sequence, so that the first region of the guide RNA can base pair with the target site. In some cases, the first region of the guide RNA may include from 10 or from about 10 nucleotides to 25 nucleotides (i.e., 10 nucleotides 25 to nucleotides; or about 10 nucleotides to about 25 nucleotides; or 10 nucleotides to about 25 nucleotides; or about 10 nucleotides to 25 nucleotides) or more. For example, the length of the base pairing region between the first region of the guide RNA and the target site in the chromosome sequence can be or can be about 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 22, 23, 24, 25 or more nucleotides. Occasionally, the length of the first region of the guide RNA can be or can be about 19, 20, or 21 nucleotides.

[0307] The guide RNA or guide polynucleotide may also include a second district, which forms a secondary structure. For example, the secondary structure generated by the guide RNA may include a stem (or hairpin) and a loop. The length of the loop and the stem may vary. For example, the length of the loop may be from or from the range of about 3 to 10 nucleotides, and the length of the stem may be from or from the range of about 6 to 20 nucleotides. The stem may include one or more protrusions of 1 to 10 or about 10 nucleotides. The total length of the second district may be from or from the range of about 16 to 60 nucleotides. For example, the length of the loop may be or may be about 4 nucleotides, and the stem may be or may be about 12 base pairs.

[0308] The guide RNA or guide polynucleotide may also include a third region at the 3' end, which may be primarily single-stranded. For example, the third region may occasionally be non-complementary to any chromosomal sequence in the cell of interest, and occasionally be non-complementary to the remainder of the guide RNA. Again, the length of the third region may vary. The length of the third region may be more than or more than about 4 nucleotides. For example, the length of the third region may be from or in the range of about 5 to 60 nucleotides.

[0309] A guide RNA or guide polynucleotide can target any exon or intron of a gene target. In some cases, a guide can target exon 1 or 2 of a gene; in other cases, a guide can target exon 3 or 4 of a gene. A composition can include multiple guide RNAs that all target the same exon, or in some cases, multiple guide RNAs can target the same exon. Exons and introns of a gene can be targeted.

[0310] The guide RNA or guide polynucleotide can target a nucleic acid sequence of 20 or about 20 nucleotides. The target nucleic acid can be less than or less than about 20 nucleotides. The target nucleic acid can be at least or at least about 5, 10, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 30, or any length between 1-100 nucleotides in length. The target nucleic acid can be at most or at most about 5, 10, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 30, 40, 50, or any length between 1-100 nucleotides in length. The target nucleic acid sequence can be about 20 bases 5' of the first nucleotide immediately adjacent to the PAM. The guide RNA can target a nucleic acid sequence. A target nucleic acid can be at least or about at least 1-10, 1-20, 1-30, 1-40, 1-50, 1-60, 1-70, 1-80, 1-90, or 1-100 nucleotides in length.

[0311] Guide Polynucleotides For example, guide RNA can refer to a nucleic acid that is capable of hybridizing to another nucleic acid, for example, a target nucleic acid or a protospacer in the genome of a cell. The guide polynucleotide can be RNA. The guide polynucleotide can be DNA. The guide polynucleotide can be programmed or configured to site-specifically bind to a nucleic acid sequence. The guide polynucleotide can include one polynucleotide chain, and can be referred to as a single guide polynucleotide. The guide polynucleotide can include two polynucleotide chains, and can be referred to as a double guide polynucleotide. The guide RNA can be introduced into a cell or embryo as an RNA molecule. For example, the RNA molecule can be transcribed in vitro and / or can be chemically synthesized. The RNA can be transcribed from a synthetic DNA molecule, for example Gene fragment. The guide RNA can then be introduced into the cell or embryo as an RNA molecule. The guide RNA can also be introduced into the cell or embryo in the form of a non-RNA nucleic acid molecule (e.g., a DNA molecule). For example, the DNA encoding the guide RNA can be operably linked to a promoter control sequence for expressing the guide RNA in the cell or embryo of interest. The RNA coding sequence can be operably linked to a promoter sequence, which is recognized by RNA polymerase III (Pol III). Plasmid vectors that can be used to express guide RNA include, but are not limited to, px330 vectors and px333 vectors. In some cases, a plasmid vector (e.g., a px333 vector) may contain at least two guide RNA encoding DNA sequences.

[0312] Methods for selecting, designing, and validating guide polynucleotides, such as guide RNAs, and targeting sequences are described in the present disclosure and are known to those skilled in the art. For example, to minimize the effects of potential enzyme substrate promiscuity of a deaminase domain (e.g., an AID domain) in a nucleoside base editor system, the number of residues that may be unintentionally targeted for deamination (e.g., off-target C residues on ssDNA that may reside inside the target nucleic acid locus) can be minimized. In addition, software tools can be used to optimize gRNAs corresponding to target nucleic acid sequences, for example, to minimize total off-target activity across the genome. For example, for each possible target domain selection using Streptococcus pyogenes Cas9, all off-target sequences (the above-selected PAMs, such as NAG or NGG) can be identified across the genome, containing up to a certain number (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10) of mismatched base pairs. The first region of the gRNA complementary to the target site can be identified, and all first regions (e.g., crRNAs) can be ranked according to their predicted off-target total score; the highest ranked target domains represent those that are likely to have the greatest on-target activity and the least off-target activity. Candidate gRNAs can be evaluated using methods known in the industry and / or as described herein.

[0313] As a non-limiting example, the target DNA hybridization sequence used in the crRNA of the guide RNA for use with Cas9 can be identified using a DNA sequence search algorithm. gRNA design can be based on the public tool sequence alignment programming (casoffinder) (as described in Bae S., Park J., & Kim J.-S.Cas-OFFinder: "A fast and versatile algorithm that searches for potential off-target sites of Cas9RNA-guidedendonucleases". Bioinformatics 30, 1473-1475 (2014)), using customer gRNA design. After calculating its whole genome off-target tendency, it is guided by this software score. Typically, the match range from perfect match to 7 mismatches is considered as a guide with a length range of 17 to 24. Once the off-target site is determined by the operation, the cumulative score is calculated for each guide and output using a web interface summary list. In addition to identifying potential target sites adjacent to the PAM sequence, the software also identifies all PAM-adjacent sequences that differ from the selected target site by 1, 2, 3, or more than 3 nucleotides. The genomic DNA sequence for the target nucleic acid sequence, such as the target gene, can be obtained and the repeats can be screened using publicly available tools such as Repeatmasker. Repeatmasker searches for repeats and low complexity regions in the input DNA sequence. The output is a detailed annotation of the repeat sequences present in the specified query sequence.

[0314] After identification, the first region of the guide RNA, such as crRNA, can be ranked in layers based on its distance from the target site, its irrelevance, and the presence of a 5' nucleotide that closely matches a relevant PAM sequence (e.g., 5'G, based on recognition of a close match in the human genome containing a relevant PAM such as the NGG PAM of S. pyogenes, the NNGRRT or NNGRRV PAM of S. aureus). As used in the present disclosure, irrelevance refers to the number of sequences in the human genome that contain the minimum number of mismatches with the target sequence. "High irrelevance" or "good irrelevance" can, for example, refer to a 20-mer target domain that does not contain identical sequences in the human genome other than the intended target, nor does it contain any sequence that contains one or two mismatches in the target sequence. Domains targeted to have good irrelevance can be selected to minimize off-target DNA cleavage.

[0315] In some embodiments, the reporting subsystem can be used to detect base editing activity and test candidate guide polynucleotides. In some embodiments, the reporting subsystem may include an analytical test based on a reporting subgene, in which the base editing activity leads to the expression of the reporting subgene. For example, the reporting subsystem may include a reporting subgene, which includes a deactivated start codon, such as a mutation on a template strand of 3'-TAC-5' to 3'-CAC-5'. When the target C is successfully deaminated, the corresponding mRNA will be transcribed into 5'-AUG-3', rather than 5'-GUG-3', so that the reporting subgene can be translated. Suitable reporting subgenes will be obvious to those skilled in the art. Non-limiting examples of reporting subgenes include genes encoding green fluorescent protein (GFP), red fluorescent protein (RFP), luciferase, secreted alkaline phosphatase (SEAP), or any other gene, whose expression is detectable and obvious to those skilled in the art. The reporting subsystem can be used to test many different gRNAs in order to determine which residue(s) will be targeted by individual deaminases relative to the target DNA sequence. sgRNAs targeting non-template strands can also be tested in order to evaluate the off-target effects of specific base editing proteins, such as Cas9 deaminase fusion proteins. In some embodiments, these gRNAs can be designed so that the mutated start codon will not base pair with the gRNA. The guide polynucleotide may include standard ribonucleotides, modified ribonucleotides (e.g., pseudouridine), ribonucleotide isomers, and / or ribonucleotide analogs. In some embodiments, the guide polynucleotide may include at least one detectable label. The detectable label can be a fluorescent group (e.g., FAM, TMR, Cy3, Cy5, Texas Red, Oregon Green, Alexa Fluors, Halo tags, or suitable fluorescent dyes), a detection label (e.g., biotin, digoxigenin, etc.), a quantum particle, or a gold particle.

[0316] The guide polynucleotide can be chemically synthesized, enzymatically synthesized, or a combination thereof. For example, the guide RNA can be synthesized using a standard phosphoramidite-based solid phase synthesis method. In addition, the guide RNA can be synthesized in vitro by operably linking the DNA encoding the guide RNA to a promoter control sequence (which is recognized by a bacteriophage RNA polymerase). Examples of suitable phage promoter sequences include T7, T3, SP6 promoter sequences, or variants thereof. In embodiments where the guide RNA comprises two separate molecules (e.g., crRNA and tracr RNA), the crRNA can be chemically synthesized, and the tracr RNA can be enzymatically synthesized.

[0317] In some embodiments, the base editor system may include a plurality of guide polynucleotides, such as gRNAs. For example, the gRNAs may target one or more target loci (e.g., at least 1 gRNA, at least 2 gRNAs, at least 5 gRNAs, at least 10 gRNAs, at least 20 gRNAs, at least 30 g RNAs, at least 50 gRNAs) included in the base editor system. The plurality of gRNA sequences may be arranged in series and preferably separated by direct repeat sequences.

[0318] The DNA sequence encoding the guide RNA or guide polynucleotide can also be part of the vector. In addition, the vector can contain additional expression control sequences (e.g., enhancer sequences, Kozak sequences, polyadenylation sequences, transcription termination sequences, etc.), selectable marker sequences (e.g., GFP or antibiotic resistance genes such as puromycin), replication origins, etc. The DNA sequence encoding the guide RNA can also be linear. The DNA sequence encoding the guide RNA or guide polynucleotide can also be circular.

[0319] In some embodiments, one or more components of the base editor system can be encoded by a DNA sequence. Such DNA sequences can be introduced into an expression system such as a cell together or separately. For example, a DNA sequence encoding a polynucleotide programmable nucleotide binding domain and a guide RNA can be introduced into a cell, and each DNA sequence can be a separate molecular component (for example, one vector contains a polynucleotide programmable nucleotide binding domain coding sequence, and a second vector contains a guide RNA coding sequence); or both can be the same molecular component (for example, a vector contains coding (and regulatory) sequences for both a polynucleotide programmable nucleotide binding domain and a guide RNA).

[0320] The guide polynucleotide may comprise one or more modifications to provide novel or enhanced characteristics to the nucleic acid. The guide polynucleotide may comprise a nucleic acid affinity tag. The guide polynucleotide may comprise synthetic nucleotides, synthetic nucleotide analogs, nucleotide derivatives, and / or modified nucleotides,

[0321] In some cases, the gRNA or guide polynucleotide may include a modification. The modification may be made at any position of the gRNA or guide polynucleotide. More than one modification may be made to a single gRNA or guide polynucleotide. The gRNA or guide polynucleotide may be subjected to quality control after modification. In some cases, quality control may include PAGE, HPLC, MS, or any combination thereof.

[0322] Modification of the gRNA or guide polynucleotide can be substitution, insertion, deletion, chemical modification, physical modification, stabilization, purification, or any combination thereof.

[0323] The gRNA or guide polynucleotide can also be modified by: 5' adenylation, 5' guanosine triphosphate end cap, 5' N7-methylguanosine triphosphate end cap, 5' triphosphate end cap, 3' phosphate, 3' phosphorothioate, 5' phosphate, 5' phosphorothioate, cis-trans thymidine dimer, trimer, C12 spacer, C3 spacer, C6 spacer, dSpacer, PC spacer, rSpacer, Spacer 18, Spacer 9, 3'-3' modification, 5'-5' modification, abasic site, acridine, azobenzene, biotin, biotin BB, biotin TEG, cholesteryl TEG, desthiobiotin TEG, DNP TEG, DNP-X, DOTA, dT-biotin, bibiotin, PC biotin, psoralen C2, psoralen C6, TINA, 3'DABCYL, black hole quencher 1, black hole quencher 2, DABCYL SE, dT-DABCYL, IRDye QC-1, QSY-21, QSY-35, QSY-7, QSY-9, a carboxyl linker, a thiol linker, a 2'-deoxyribonucleoside analog purine, a 2'-deoxyribonucleoside analog pyrimidine, a ribonucleoside analog, a 2'-O-methyl ribonucleoside analog, a sugar modification analog, a wobble / universal base, a fluorescent dye label, 2'-fluoro RNA, 2'-O-methyl RNA, methyl phosphonate, phosphodiester DNA, phosphodiester RNA, phosphorothioate DNA, phosphorothioate RNA, UNA, pseudouridine-5'-triphosphate, 5'-methylcytidine-5'-triphosphate, or any combination thereof.

[0324] In some cases, the modification is permanent. In other cases, the modification is temporary. In some cases, multiple modifications can be made to the gRNA or guide polynucleotide. gRNA or guide polynucleotide modifications can change the physicochemical properties of the nucleotides, such as their conformation, polarity, water repellency, chemical reactivity, base pairing interactions, or any combination thereof.

[0325] The modification can also be a phosphorothioate substitution. In some cases, the natural phosphodiester bond may be susceptible to rapid degradation by cellular nucleases, and modifications using nucleotide linkages substituted with phosphorothioate (PS) bonds may be more stable to hydrolysis by cellular degradation. Modifications can improve the stability of the gRNA or guide polynucleotide. Modifications can also enhance biological activity. In some cases, the RNA gRNA enhanced with phosphorothioate can inhibit RNase A, RNase T1, fetal bovine serum nuclease, or any combination thereof. These properties allow the use of PS-RNA gRNAs in applications where there is a high probability of exposure to nucleases in vivo or in vitro. For example, phosphorothioate (PS) bonds can be introduced between the last 3-5 nucleotides of the 5'- or "-end of the gRNA, which can inhibit exonuclease degradation. In some cases, phosphorothioate bonds can be added throughout the gRNA to reduce attack by endonucleases.

[0326] Protospacer adjacent motif

[0327] The term "protospacer adjacent motif (PAM)" or PAM-like motif refers to the 2-6 base pair DNA sequence immediately following the DNA sequence targeted by the Cas9 nuclease in the CRISPR bacterial adaptive immune system. In some embodiments, the PAM can be a 5' PAM (i.e., located upstream of the 5' end of the protospacer). In other embodiments, the PAM can be a 3' PAM (i.e., located downstream of the 5' end of the protospacer).

[0328] A protospacer adjacent motif (PAM) or PAM-like motif refers to a 2-6 base pair DNA sequence immediately following a DNA sequence targeted by the Cas9 nuclease in the CRISPR bacterial adaptive immune system. In some embodiments, the PAM can be a 5'PAM (i.e., located upstream of the 5' end of the protospacer). In other embodiments, the PAM can be a 3'PAM (i.e., located downstream of the 5' end of the protospacer). The PAM sequence is required for target binding, but the exact sequence depends on the type of Cas protein. The base editors provided in the present disclosure may include a CRISPR protein-derived domain that can bind to a nucleotide sequence containing a canonical or non-canonical protospacer adjacent motif (PAM) sequence. The PAM site is a nucleotide sequence adjacent to a target polynucleotide sequence. Several aspects of the present disclosure provide base editors that include all or part of a CRISPR protein with different PAM specificities. For example, a typical Cas9 protein, such as Cas9 from Streptococcus pyogenes (spCas9), requires a canonical NGG PAM sequence to bind to a specific nucleic acid region, where the N in NGG is adenine (A), thymine (T), guanine (G), or cytosine (C), and G is guanine. The PAM can be CRISPR protein specific and can be different between different base editors comprising different CRISPR protein-derived domains. The PAM can be 5' or 3' of the target sequence. The PAM can be upstream or downstream of the target sequence. The PAM can be 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more nucleotides in length. Often the PAM is between 2 and 6 nucleotides in length.

[0329] In some embodiments, the Cas9 domain is a Cas9 domain from Streptococcus pyogenes (SpCas9). In some embodiments, the SpCas9 domain is a nuclease active SpCas9, a nuclease inactive SpCas9 (SpCas9d), or a SpCas9 nickase (SpCas9n). In some embodiments, SpCas9 comprises a D9X mutation, or a corresponding mutation in any of the amino acid sequences provided in the present disclosure, wherein X is any amino acid (but excluding D). In some embodiments, SpCas9 comprises a D9A mutation, or a corresponding mutation in any of the amino acid sequences provided in the present disclosure. In some embodiments, the SpCas9 domain, SpCas9d domain, or SpCas9n domain can bind to a nucleic acid sequence having a non-canonical PAM. In some embodiments, the SpCas9 domain, SpCas9d domain, or SpCas9n domain can bind to a nucleic acid sequence having an NGG, NGA, or NGCG PAM sequence. In some embodiments, the SpCas9 domain comprises one or more of D1135X, R1335X, and T1337X mutations, or corresponding mutations in any of the amino acid sequences provided in the present disclosure, wherein X is any one amino acid. In some embodiments, the SpCas9 domain comprises one or more of D1135E, R1335Q, and T1337R mutations, or corresponding mutations in any of the amino acid sequences provided in the present disclosure. In some embodiments, the SpCas9 domain comprises one or more of D1135E, R1335Q, and T1337R mutations, or corresponding mutations in any of the amino acid sequences provided in the present disclosure. In some embodiments, the SpCas9 domain comprises one or more of D1135X, R1335X, and T1337X mutations, or corresponding mutations in any of the amino acid sequences provided in the present disclosure, wherein X is any one amino acid. In some embodiments, the SpCas9 domain comprises one or more of D1135V, R1335Q, and T1337R mutations, or corresponding mutations in any of the amino acid sequences provided in the present disclosure. In some embodiments, the SpCas9 domain comprises one or more of D1135V, R1335Q, and T1337R mutations, or corresponding mutations in any of the amino acid sequences provided in the present disclosure. In some embodiments, the SpCas9 domain comprises one or more of D1135X, G1218X, R1335X, and T1337X mutations, or corresponding mutations in any of the amino acid sequences provided in the present disclosure, wherein X is any amino acid. In some embodiments, the SpCas9 domain comprises one or more of D1135V, G1218R, R1335Q, and T1337R mutations, or corresponding mutations in any of the amino acid sequences provided in the present disclosure.In some embodiments, the SpCas9 domain comprises one or more of D1135V, G1218R, R1335Q, and T1337R mutations, or the corresponding mutations in any of the amino acid sequences provided in the present disclosure.

[0330] In some embodiments, the Cas9 domain of any of the fusion proteins provided in the present disclosure comprises an amino acid sequence that is at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or at least 99.5% identical to a Cas9 polypeptide described herein. In some embodiments, the Cas9 domain of any of the fusion proteins provided in the present disclosure comprises an amino acid sequence of any Cas9 polypeptide described herein. In some embodiments, the Cas9 domain of any of the fusion proteins provided in the present disclosure consists of an amino acid sequence of any Cas9 polypeptide described herein.

[0331] The amino acid sequence of an embodiment of a SpCas9 protein capable of binding to a PAM sequence is as follows.

[0332] The amino acid sequence of an example of SpCas9 bound to a PAM is as follows:

[0333]

[0334]

[0335] The amino acid sequence of an embodiment of SpCas9n bound to a PAM is as follows:

[0336]

[0337] The amino acid sequence of an example of SpEQR Cas9 bound to a PAM is as follows:

[0338] In this sequence, residues E1135, Q1335, and R1337, which can be mutated from D1135, R1335, and T1337 to obtain SpEQR Cas9, are underlined and in bold.

[0339] The amino acid sequence of an example of SpVQR Cas9 bound to a PAM is as follows:

[0340]

[0341] In this sequence, residues V1135, Q1335, and R1337, which can be mutated from D1135, R1335, and T1337 to obtain SpVQR Cas9, are underlined and in bold.

[0342] The amino acid sequence of an example of SpVRER Cas9 bound to a PAM is as follows:

[0343]

[0344]

[0345] The amino acid sequence of an example of SpVRQR Cas9 bound to a PAM is as follows:

[0346]

[0347] As above, residues V1135, R1218, Q1335, and R1337, which can be obtained from mutations of D1135, G1218, R1335, and T1337 to obtain SpVRQR Cas9, are underlined and in bold.

[0348] In some embodiments, the Cas9 domain is a recombinant Cas9 domain. In some embodiments, the recombinant Cas9 domain is a SpyMacCas9 domain. In some embodiments, the SpyMacCas9 domain is a nuclease active SpyMacCas9, a nuclease inactive SpyMacCas9 (SpyMacCas9d), or a SpyMacCas9 nickase (SpyMacCas9n). In some embodiments, the SaCas9 domain, SaCas9d domain, or SaCas9n domain can bind to a nucleic acid sequence with a non-canonical PAM. In some embodiments, the SaCas9 domain, SaCas9d domain, or SaCas9n domain can bind to a nucleic acid sequence with a NAA PAM sequence.

[0349] SpyMacCas9 Example

[0350]

[0351] High-fidelity Cas9 domain

[0352] Several aspects disclosed herein provide high-fidelity Cas9 domains. In several embodiments, the high-fidelity Cas9 domain is a genetically modified Cas9 domain comprising one or more mutations, which, compared to the corresponding wild-type Cas9 domain, reduces the electrostatic interaction between the Cas9 domain and the sugar-phosphate backbone of DNA. Without being bound by any particular theory, a high-fidelity Cas9 domain with reduced electrostatic interaction with the sugar-phosphate backbone of DNA may have fewer off-target effects. In several embodiments, the Cas9 domain (e.g., a wild-type Cas9 domain) comprises one or more mutations that reduce the electrostatic interaction between the Cas9 domain and the sugar-phosphate backbone of DNA. In some embodiments, the Cas9 domain comprises one or more mutations that reduce the association of the Cas9 domain with the sugar-phosphate backbone of DNA by at least 1%, at least 2%, at least 3%, at least 4%, at least 5%, at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, or at least 70%.

[0353] In some embodiments, any of the Cas9 fusion proteins provided in the present disclosure comprises one or more of N497X, R661X, Q695X, and / or Q926X mutations, or corresponding mutations in any of the amino acid sequences provided in the present disclosure, wherein X is any amino acid. In some embodiments, any of the Cas9 fusion proteins provided in the present disclosure comprises one or more of N497A, R661A, Q695A, and / or Q926A mutations, or corresponding mutations in any of the amino acid sequences provided in the present disclosure. In some embodiments, any of the Cas9 fusion proteins provided in the present disclosure comprises a D10A mutation, or corresponding mutations in any of the amino acid sequences provided in the present disclosure. For example, Cas9 domains with high fidelity have been described in Kleinstiver, BP, et al. "High-fidelity CRISPR-Cas9 nucleases with no detectable genome-wide off-target effects." Nature 529, 490-495 (2016); and Slaymaker, IM, et al. "Rationally engineered Cas9 nucleases with improved specificity." Science 351, 84-88 (2015), each of which is incorporated by reference into the disclosure of this specification.

[0354] Compared to Cas9, high-fidelity Cas9 domain mutations are shown in bold and underlined.

[0355]

[0356]

[0357] In some cases, the variant Cas9 protein has H840A, P475A, W476A, N477A, ​​D1125A, W1126A, and D1127A mutations, so that the polypeptide has a reduced ability to cleave target DNA or RNA. Such Cas9 proteins have a reduced ability to cleave target DNA (e.g., single-stranded target DNA), but retain the ability to bind to target DNA (e.g., single-stranded target DNA). As another non-limiting example, in some cases, the variant Cas9 protein has D10A, H840A, P475A, W476A, N477A, ​​D1125A, W1126A, and D1127A mutations, so that the polypeptide has a reduced ability to cleave target DNA or RNA. Such Cas9 proteins have a reduced ability to cleave target DNA (e.g., single-stranded target DNA), but retain the ability to bind to target DNA (e.g., single-stranded target DNA). In some cases, when the variant Cas9 protein has W476A and W1126A mutations, or when the variant Cas9 protein has P475A, W476A, N477A, ​​D1125A, W1126A, and D1127A mutations, the variant Cas9 protein cannot effectively bind to the PAM sequence. Therefore, in some of these cases, when such variant Cas9 proteins are used in a binding method, the method does not require a PAM sequence. In other words, in some cases, when such variant Cas9 proteins are used in a binding method, the method may include a guide RNA, but the method may be performed in the absence of a PAM sequence (and therefore, the binding specificity is provided by the targeting segment of the guide RNA). Other residues can be mutated to achieve the above effects (i.e., inactivate one or the other nuclease portion). As non-limiting examples, residues D10, G12, G17, E762, H840, N854, N863, H982, H983, A984, D986, and / or A987 may be mutated (i.e., substituted). Again, mutations other than alanine substitutions are suitable.

[0358] In some embodiments, the CRISPR protein-derived domain of the base editor may include all or part of a Cas9 protein with a canonical PAM sequence (NGG). In other embodiments, the CRISPR protein-derived domain of the base editor may employ a non-canonical PAM sequence. Such sequences have been described in the industry and will be apparent to those skilled in the art. For example, Cas9 domains that bind to non-canonical PAM sequences have been described in Kleinstiver, BP, et al., "Engineered CRISPR-Cas9 nucleases with altered PAM specificities" Nature 523, 481-485 (2015); and Kleinstiver, BP, et al., "Broadening the targeting range of Staphylococcus aureus CRISPR-Cas9 by modifying PAM recognition" Nature Biotechnology 33, 1293-1298 (2015), each of which is incorporated by reference into the disclosure of this specification in its entirety.

[0359] In some embodiments, a PAM recognized by a CRISPR protein-derived domain of a base editor can be provided to a cell on a separate oligonucleotide to an insert encoding a base editor (e.g., an AAV insert). In such cases, the PAM provided on the separate oligonucleotide can allow for cleavage of a target sequence that would otherwise not be cleaved because no adjacent PAM is present on the same polynucleotide of the target sequence.

[0360] In one embodiment, Streptococcus pyogenes Cas9 (SpCas9) can be used as a CRISPR nuclease for genome modification. However, others can also be used. In some cases, different nucleases can be used to target certain genome targets. In some cases, synthetic SpCas9 derivative variants that do not contain the NGG PAM sequence can be used. In addition, other Cas9 analogs from various species have been identified, and these "SpCas9" can be combined with various PAM sequences, which are also useful in the disclosure herein. For example, the relatively large size of SpCas9 (about 4kb coding sequence) also obtained SpCas9 cDNA that cannot be effectively expressed in cells. In contrast, the coding sequence of Staphylococcus aureus Cas9 (SaCas9) is about 1 kilobase (kb) shorter than SpCas9, which may allow it to be effectively expressed in cells. Similar to SpCas9, SaCas9 endonucleases can modify target genes in vitro in mammalian cells and in vivo in mice. In some cases, Cas proteins can target different PAM sequences. In some cases, the target gene can be adjacent to the Cas9 PAM, such as 5'-NGG. In other cases, other Cas9 species have different PAM requirements. For example, other PAMs such as Streptococcus thermophilus (5'-NNAGAA for CRISPR1 and 5'-NGGNG for CRISPR3) and Neisseria meningitidis (5'-NNNNGATT) are also found adjacent to target genes.

[0361] In some embodiments, for the S. pyogenes system, the target gene sequence can be in front of (i.e., at the 5') of the 5'-NGG PAM, and the 20-nt guide RNA sequence can be base paired with the opposite strand to mediate Cas9 cleavage adjacent to the PAM. In some cases, the adjacent cut can be or can be about 3 base pairs upstream of the PAM. In some cases, the adjacent cut can be or can be about 10 base pairs upstream of the PAM. In some cases, the adjacent cut can be or can be about 0-20 base pairs upstream of the PAM. For example, the adjacent cut can be immediately upstream of the PAM, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30 base pairs. The adjacent cut can also be 1 to 30 base pairs downstream of the PAM.

[0362] Fusion proteins containing a nuclear localization sequence (NLS)

[0363] Vectors encoding CRISPR enzymes comprising one or more nuclear localization sequences (NLS) may be used. For example, about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 NLSs may be used or may be used. The CRISPR enzyme may comprise an NLS at or near the amino terminus, about or more than about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 NLSs at or near the carboxy terminus, or any combination thereof (e.g., one or more NLSs at the amino terminus, and one or more NLSs at the carboxy terminus). When more than one NLS is present, each may be independently selected so that a single NLS may be present in more than one copy, and / or in combination with one or more other NLSs to be present in more than one copy.

[0364] The CRISPR enzyme used in the method may comprise about 6 NLSs. An NLS is considered to be near the N-terminus or C-terminus when the amino acid closest to the NLS is within about 50 amino acids along the polypeptide chain from the N-terminus or C-terminus, e.g., within 1, 2, 3, 4, 5, 10, 15, 20, 25, 30, 40, or 50 amino acids.

[0365] In some embodiments, the NLS comprises the amino acid sequence PKKKRKVEGADKRTADGSEFES PKKKRKV, KRTADGSEFESPKKKRKV, KRPAATKKAGQAKKKK, KKTELQTTNAENKTKL, KRGINDRNFWRGENGRKTR, RKSGKIAAIVVKRPRKPKKKRKV, or MDSLLMNRRKFLYQFKNVRWAKGRRETYLC.

[0366] In some embodiments, the NLS is present in a linker, or the NLS is flanked by a linker, e.g., a linker described herein. In some embodiments, the N-terminal or C-terminal NLS is a two-part NLS. A two-part NLS comprises two basic amino acid clusters separated by a relatively short spacer sequence (hence a two-part -2 part, whereas a one-part NLS does not). The NLS of nucleoplasmin, KR[PAATKKAGQA]KKKK, is a prototype of a ubiquitous bipartite signal: two basic amino acid clusters separated by a spacer of about 10 amino acids. The sequence of an example of a two-part NLS is as follows:

[0367] PKKKRKVEGADKRTADGSEFES PKKKRKV.

[0368] In some embodiments, the fusion proteins of the invention do not include a spacer sequence. In some embodiments, there is a spacer sequence between one or more of the domains or proteins.

[0369] The PAM sequence can be any PAM sequence known in the art. Suitable PAM sequences include, but are not limited to, NGG, NGA, NGC, NGN, NGT, NGCG, NGAG, NGAN, NGNG, NGCN, NGCG, NGTN, NNGRRT, NNNRRT, NNGRR(N), TTTV, TYCV, TYCV, TATV, NNNNGATT, NNAGAAW, or NAAAC. Y is pyrimidine; N is any nucleotide base; and W is A or T.

[0370] Nucleoside base editing domain

[0371] Described herein are base editors comprising a fusion protein that includes a polynucleotide programmable nucleotide binding domain and a nucleoside base editing domain (e.g., a deaminase domain). The base editor can be programmed to edit one or more bases in a target polynucleotide sequence through interaction with a guide polynucleotide that can recognize the target sequence. Once the target sequence has been identified, the base editor is anchored to the polynucleotide to be edited, and then the deaminase domain component of the base editor can edit the target base.

[0372] In some embodiments, the nucleoside base editing domain is a deaminase domain. In some cases, the deaminase domain can be a cytosine deaminase or a cytidine deaminase. In some embodiments, "cytosine deaminase" or "cytidine deaminase" can be used interchangeably. In some cases, in some cases, the deaminase domain can be an adenine deaminase or an adenosine deaminase. In some embodiments, "adenine deaminase" or "adenosine deaminase" can be used interchangeably. Details of nucleoside base editing proteins are described in International PCT Application Nos. PCT / 2017 / 045381 (WO2018 / 027078) and PCT / US2016 / 058344 (WO2017 / 070632), each of which is incorporated by reference into the disclosure of this specification in its entirety. Please also refer to Komor, AC, et al., "Programmable editing of target bases in genomic DNA without double-stranded DNA cleavage," Nature 533, 420-424 (2016); Gaudelli, NM, et al., "Programmable base editing of A·T to G·C in genomic DNA without DNA cleavage," Nature 551, 464-471 (2017); and Komor, AC, et al., "Improved base excision repair inhibition and bacteriophage μGam protein with higher efficiency and product purity to obtain C:G-to-T:A base editor," Science Advances 3:eaao4774 (2017), the entire contents of which are incorporated by reference into the disclosure of this specification.

[0373] C to T Edit

[0374] In some embodiments, base editors disclosed herein comprise a fusion protein comprising a cytidine deaminase domain that is capable of deaminating a target cytidine (C) base of a polynucleotide to produce uridine (U), which has the base pairing properties of thymine. In some embodiments, for example, where the polynucleotide is double-stranded (e.g., DNA), the uridine base can then be substituted with a thymidine base (e.g., by a cellular repair mechanism) to obtain a C:G to T:A conversion. In other embodiments, deamination of C to U by a base editor in a nucleic acid may not be accompanied by a U to T substitution.

[0375] Deamination of a target C in a polynucleotide to obtain U is a non-limiting example of a type of base editing that can be performed by a base editor described herein. In another embodiment, a base editor comprising a cytidine deaminase domain can mediate the conversion of a cytosine (C) base to a purine (G) base. For example, the U of a polynucleotide produced by deaminating cytidine by the cytidine deaminase domain of a base editor can be excised from the polynucleotide by a base excision repair mechanism (e.g., by a uracil DNA glycosylase (UDG) domain) to produce an abasic site. Then, the nucleoside base relative to the abasic site can be replaced by another base (such as C) by, for example, a translesion polymerase (e.g., by a cell repair mechanism). Although the nucleoside base relative to the abasic site is typically replaced by C, other substitutions (e.g., A, G, or T) may also occur.

[0376] Thus, in some embodiments, the base editors described herein comprise a deaminase domain (e.g., a cytidine deaminase domain) that is capable of deaminating a target C in a polynucleotide to a U. Furthermore, as described in detail below, the base editor may comprise an additional domain that, in some embodiments, assists in converting the deaminated U to a T or a G. For example, a base editor comprising a cytidine deaminase domain may further comprise a uracil glycosylase inhibitor (UGI) domain to mediate the replacement of U with T, completing a C to T base editing event. In another embodiment, a base editor may be combined with a translesion polymerase to improve the efficiency of C to G base editing because the translesion polymerase can assist in binding C relative to the abasic site (i.e., resulting in G binding to the abasic site, completing a C to G base editing event).

[0377] Base editors comprising a cytidine deaminase as a domain can deaminate a target C in any polynucleotide, including DNA, RNA, and DNA-RNA hybrids. Typically, a cytidine deaminase catalyzes a C nucleobase located in a single-stranded portion of a polynucleotide. In several embodiments, the entire polynucleotide comprising the target C can be single-stranded. For example, a cytidine deaminase incorporated into a base editor can deaminate a target C in a single-stranded RNA polynucleotide. In other embodiments, a base editor comprising a cytidine deaminase domain can act on a double-stranded polynucleotide, but the target C can be located in a portion of a polynucleotide that is in a single-stranded state during the deamination reaction. For example, in an embodiment in which the NAGPB domain comprises a Cas9 domain, when a Cas9-gRNA-target DNA complex is generated, several nucleotides can be left unpaired, resulting in the formation of a Cas9 "R-loop complex". These unpaired nucleotides can generate single-stranded DNA bubbles, which can serve as enzyme substrates for single-stranded specific nucleotide deaminase enzymes (e.g., cytidine deaminase).

[0378] In some embodiments, the cytidine deaminase of the base editor may include all or part of the apolipoprotein B mRNA editing complex (APOBEC) family deaminase. APOBEC is an evolutionarily conserved cytidine deaminase family. Members of this family are C to U editing enzymes. The N-terminal domain of the APOBEC protein is a catalytic domain, and the C-terminal domain is a pseudocatalytic domain. More specifically, the catalytic domain is a zinc-dependent cytidine deaminase domain and is important for cytidine deamination. APOBEC family members include APOBEC1, APOBEC2, APOBEC3A, APOBEC3B, APOBEC3C, APOBEC3D ("APOBEC3E" now refers to this), APOBEC3F, APOBEC3G, APOBEC3H, APOBEC4, and activation-induced (cytidine) deaminase. In some embodiments, the deaminase incorporated into the base editor includes all or part of the APOBEC1 deaminase. In some embodiments, the deaminase incorporated into the base editor comprises all or part of an APOBEC2 deaminase. In some embodiments, the deaminase incorporated into the base editor comprises all or part of an APOBEC3 deaminase. In some embodiments, the deaminase incorporated into the base editor comprises all or part of an APOBEC3A deaminase. In some embodiments, the deaminase incorporated into the base editor comprises all or part of an APOBEC3B deaminase. In some embodiments, the deaminase incorporated into the base editor comprises all or part of an APOBEC3C deaminase. In some embodiments, the deaminase incorporated into the base editor comprises all or part of an APOBEC3D deaminase. In some embodiments, the deaminase incorporated into the base editor comprises all or part of an APOBEC3E deaminase. In some embodiments, the deaminase incorporated into the base editor comprises all or part of an APOBEC3F deaminase. In some embodiments, the deaminase incorporated into the base editor comprises all or part of an APOBEC3G deaminase. In some embodiments, the deaminase incorporated into the base editor comprises all or part of an APOBEC3H deaminase. In some embodiments, the deaminase incorporated into the base editor comprises all or part of an APOBEC4 deaminase. In some embodiments, the deaminase incorporated into the base editor comprises all or part of an activation-induced deaminase (AID). In some embodiments, the deaminase incorporated into the base editor comprises all or part of cytidine deaminase 1 (CDA1). It should be understood that the base editor may comprise a deaminase from any organism (e.g., human or rat). In some embodiments, the deaminase domain of the base editor is from a human, chimpanzee, orangutan, monkey, cow, dog, rat, or mouse. In some embodiments, the deaminase domain of the base editor is derived from a rat (e.g., rat APOBEC1). In some embodiments, the deaminase domain of the base editor is human APOBEC1.In some embodiments, the deaminase domain of the base editor is pmCDA1.

[0379] The amino acid sequence and nucleic acid sequence of pmCDA1 are shown below.

[0380] >tr|A5H718|A5H718_PETMA cytosine deaminase OS=Hamidocarpus OX=7757PE=2SV=1 amino acid sequence:

[0381]

[0382] Nucleic acid sequence: >EF094822.1 Hagfish isolate PmCDA.21 cytosine deaminase mRNA, complete cds:

[0383]

[0384] The amino acid sequence and nucleic acid sequence of the coding sequence (CDS) of human activation-induced cytidine deaminase (AID) are shown below.

[0385] >tr|Q6QJ80|Q6QJ80_HUMAN activation-induced cytidine deaminase OS=Homo sapiens OX=9606GN=AICDA PE=2SV=1 Amino acid sequence:

[0386]

[0387] Nucleotide sequence: >NG_011588.1:5001-15681 Homo sapiens activation-induced cytidine deaminase (AICDA), RefSeqGene (LRG_17) on chromosome 12:

[0388]

[0389]

[0390]

[0391]

[0392]

[0393] According to aspects of the present disclosure, other examples of deaminases that can be fused to Cas9 are provided below. It should be understood that in some embodiments, the active domain of a separate sequence can be used, for example, a domain without a localization signal (nuclear localization sequence, without a nuclear export signal, a cytoplasmic localization signal).

[0394] Human AID:

[0395]

[0396] (Lower line: nuclear localization sequence; double lower line: nuclear export signal)

[0397] Mouse AID:

[0398]

[0399] (Lower line: nuclear localization sequence; double lower line: nuclear export signal)

[0400] Canine AID:

[0401]

[0402] (Lower line: nuclear localization sequence; double lower line: nuclear export signal)

[0403] Cattle AID:

[0404]

[0405] (Lower line: nuclear localization sequence; double lower line: nuclear export signal)

[0406] Rat AID:

[0407]

[0408] (Lower line: nuclear localization sequence; double lower line: nuclear export signal)

[0409] Mouse APOBEC3:

[0410]

[0411] (italics: nucleic acid editing domain)

[0412] Rat APOBEC-3:

[0413]

[0414] (italics: nucleic acid editing domain)

[0415] Macaque APOBEC-3G:

[0416]

[0417] (Italics: nucleic acid editing domain; underlined: cytoplasmic localization signal)

[0418] Chimpanzee APOBEC-3G:

[0419]

[0420] (Italics: nucleic acid editing domain; underlined: cytoplasmic localization signal)

[0421] Green Monkey APOBEC-3G:

[0422]

[0423] (Italics: nucleic acid editing domain; underlined: cytoplasmic localization signal)

[0424] Human APOBEC-3G:

[0425]

[0426] (Italics: nucleic acid editing domain; underlined: cytoplasmic localization signal)

[0427] Human APOBEC-3F:

[0428]

[0429] (italics: nucleic acid editing domain)

[0430] Human APOBEC-3B:

[0431]

[0432] (italics: nucleic acid editing domain)

[0433] Rat APOBEC-3B:

[0434]

[0435] Bovine APOBEC-3B:

[0436]

[0437] Chimpanzee APOBEC-3B:

[0438]

[0439] Human APOBEC-3C:

[0440]

[0441] (italics: nucleic acid editing domain)

[0442] Orangutan APOBEC-3C:

[0443]

[0444] Human APOBEC-3A:

[0445]

[0446] (italics: nucleic acid editing domain)

[0447] Macaque APOBEC-3A:

[0448]

[0449] (italics: nucleic acid editing domain)

[0450] Bovine APOBEC-3A:

[0451]

[0452] (italics: nucleic acid editing domain)

[0453] Human APOBEC-3H:

[0454]

[0455] (italics: nucleic acid editing domain)

[0456] Macaque APOBEC-3H:

[0457]

[0458] Human APOBEC-3D:

[0459]

[0460] (italics: nucleic acid editing domain)

[0461] Human APOBEC-1:

[0462]

[0463] Mouse APOBEC-1:

[0464]

[0465] Rat APOBEC-1:

[0466]

[0467] Human APOBEC-2:

[0468]

[0469] Mouse APOBEC-2:

[0470]

[0471] Rat APOBEC-2:

[0472]

[0473] Bovine APOBEC-2:

[0474]

[0475] Hagfish CDA1 (pmCDA1):

[0476]

[0477] Human APOBEC3G D316R D317R:

[0478]

[0479] Human APOBEC3G chain A:

[0480]

[0481] Human APOBEC3G chain A D120R D121R:

[0482]

[0483] Some aspects of the present disclosure are based on the recognition that, for example, by making point mutations in the deaminase domain, the catalytic activity of the deaminase domain of any of the fusion proteins described herein is regulated to affect the processivity of the fusion protein (e.g., base editor). For example, a mutation that reduces (but does not eliminate) the catalytic activity of the deaminase domain inside the base editing fusion protein makes it less likely that the deaminase domain will catalyze the deamination of residues adjacent to the target residue, thereby narrowing the deamination window. The ability to narrow the deamination window can prevent residues adjacent to a specific target residue from being undesirably deaminated, which can reduce or prevent off-target effects.

[0484] For example, in some embodiments, the APOBEC deaminase incorporated into the base editor may include one or more mutations selected from the group consisting of H121X, H122X, R126X, R126X, R118X, W90X, W90X, and R132X of rAPOBEC1; or one or more corresponding mutations in another APOBEC deaminase, wherein X is any amino acid. In some embodiments, the APOBEC deaminase incorporated into the base editor may include one or more mutations selected from the group consisting of H121R, H122R, R126A, R126E, R118A, W90A, W90Y, and R132E of rAPOBEC1; or one or more corresponding mutations in another APOBEC deaminase.

[0485] For example, in some embodiments, the APOBEC deaminase incorporated into the base editor may include one or more mutations selected from the group consisting of D316X, D317X, R320X, R320X, R313X, W285X, W285X, R326X of hAPOBEC3G; or one or more corresponding mutations in another APOBEC deaminase, wherein X is any amino acid. In some embodiments, the APOBEC deaminase incorporated into the base editor may include one or more mutations selected from the group consisting of D316R, D317R, R320A, R320E, R313A, W285A, W285Y, R326E of hAPOBEC3G; or one or more corresponding mutations in another APOBEC deaminase.

[0486] In some embodiments, the APOBEC deaminase incorporated into the base editor may include the H121R and H122R mutations of rAPOBEC1; or one or more corresponding mutations in another APOBEC deaminase. In some embodiments, the APOBEC deaminase incorporated into the base editor may include the R126A mutation of rAPOBEC1; or one or more corresponding mutations in another APOBEC deaminase. In some embodiments, the APOBEC deaminase incorporated into the base editor may include the R126E mutation of rAPOBEC1; or one or more corresponding mutations in another APOBEC deaminase. In some embodiments, the APOBEC deaminase incorporated into the base editor may include the R118A mutation of rAPOBEC1; or one or more corresponding mutations in another APOBEC deaminase. In some embodiments, the APOBEC deaminase incorporated into the base editor may include the W90A mutation of rAPOBEC1; or one or more corresponding mutations in another APOBEC deaminase. In some embodiments, the APOBEC deaminase incorporated into the base editor may include the W90Y mutation of rAPOBEC1; or one or more corresponding mutations in another APOBEC deaminase. In some embodiments, the APOBEC deaminase incorporated into the base editor may include the R132E mutation of rAPOBEC1; or one or more corresponding mutations in another APOBEC deaminase. In some embodiments, the APOBEC deaminase incorporated into the base editor may include the W90Y and R126E mutations of rAPOBEC1; or one or more corresponding mutations in another APOBEC deaminase. In some embodiments, the APOBEC deaminase incorporated into the base editor may include the R126E and R132E mutations of rAPOBEC1; or one or more corresponding mutations in another APOBEC deaminase. In some embodiments, the APOBEC deaminase incorporated into the base editor may include the W90Y and R132E mutations of rAPOBEC1; or one or more corresponding mutations in another APOBEC deaminase. In some embodiments, the APOBEC deaminase incorporated into the base editor may comprise the W90Y, R126E, and R132E mutations of rAPOBEC1; or one or more corresponding mutations in another APOBEC deaminase.

[0487] In some embodiments, the APOBEC deaminase incorporated into the base editor may include the D316R and D317R mutations of hAPOBEC3G; or one or more corresponding mutations in another APOBEC deaminase. In some embodiments, the APOBEC deaminase incorporated into the base editor may include the R320A mutation of hAPOBEC3G; or one or more corresponding mutations in another APOBEC deaminase. In some embodiments, the APOBEC deaminase incorporated into the base editor may include the R320E mutation of hAPOBEC3G; or one or more corresponding mutations in another APOBEC deaminase. In some embodiments, the APOBEC deaminase incorporated into the base editor may include the R313A mutation of hAPOBEC3G; or one or more corresponding mutations in another APOBEC deaminase. In some embodiments, the APOBEC deaminase incorporated into the base editor may include the W285A mutation of hAPOBEC3G; or one or more corresponding mutations in another APOBEC deaminase. In some embodiments, the APOBEC deaminase incorporated into the base editor may include the W285Y mutation of hAPOBEC3G; or one or more corresponding mutations in another APOBEC deaminase. In some embodiments, the APOBEC deaminase incorporated into the base editor may include the R326E mutation of hAPOBEC3G; or one or more corresponding mutations in another APOBEC deaminase. In some embodiments, the APOBEC deaminase incorporated into the base editor may include the W285Y and R320E mutations of hAPOBEC3G; or one or more corresponding mutations in another APOBEC deaminase. In some embodiments, the APOBEC deaminase incorporated into the base editor may include the R320E and R326E mutations of hAPOBEC3G; or one or more corresponding mutations in another APOBEC deaminase. In some embodiments, the APOBEC deaminase incorporated into the base editor may include the W285Y and R326E mutations of hAPOBEC3G; or one or more corresponding mutations in another APOBEC deaminase. In some embodiments, the APOBEC deaminase incorporated into the base editor may include the W285Y, R320E, and R326E mutations of hAPOBEC3G; or one or more corresponding mutations in another APOBEC deaminase.

[0488] A variety of modified cytidine deaminases are commercially available, including, but not limited to, SaBE3, SaKKH-BE3, VQR-BE3, EQR-BE3, VRER-BE3, YE1-BE3, EE-BE3, YE2-BE3, and YEE-BE3 (plasmids 85169, 85170, 85171, 85172, 85173, 85174, 85175, 85176, 85177) from Addgene.

[0489] Details of the C to T nucleoside base editing protein are described in International PCT Application Nos. PCT / US2016 / 058344 (WO2017 / 070632), and Komor, AC, et al., "Programmable editing of target bases in genomic DNA without double-stranded DNA cleavage," Nature 533, 420-424 (2016), the entire contents of which are incorporated by reference into the disclosure of this specification.

[0490] A to GEdit

[0491] In some embodiments, the base editors described herein may include a deaminase domain that includes an adenosine deaminase. Such an adenosine deaminase domain of a base editor can assist in editing an A nucleobase to a G nucleobase by deaminating adenine (A) to generate inosine (I), which has the base pairing property of guanine (G). Adenosine deaminase can deaminate (i.e., remove the amino group) of adenine in a deoxyadenosine residue in deoxyribonucleic acid (DNA).

[0492] In some embodiments, the nucleoside base editors provided in the present disclosure can be manufactured by fusing one or more protein domains together to generate a fusion protein. In certain embodiments, the fusion protein provided in the present disclosure comprises one or more features that improve the base editing activity (e.g., efficiency, selectivity, and specificity) of the fusion protein. For example, the fusion protein provided in the present disclosure may include a Cas9 domain with reduced nuclease activity. In some embodiments, the fusion protein provided in the present disclosure may have a Cas9 domain without nuclease activity (dCas9), or a Cas9 domain that cuts one strand of a diploid DNA molecule, referred to as a Cas9 nickase (nCas9). Without being limited by any particular theory, the presence of a catalytic residue (e.g., H840) maintains the activity of Cas9 to cleave a non-edited (e.g., non-deamination) strand containing T relative to target A. Mutations of the catalytic residues of Cas9 (e.g., D10 to A10) prevent the cleavage of edited strands containing target A residues. These Cas9 variants can generate single-stranded DNA breaks (nicks) at specific locations based on the target sequence defined by the gRNA, leading to repair of the non-edited strand, with the end result being a T to C change on the non-edited strand. In some embodiments, the A-to-G base editor further comprises an inosine base excision repair inhibitor, such as a uracil glycosylase inhibitor (UGI) domain or a catalytically inactive inosine-specific nuclease. Without wishing to be bound by any particular theory, the UGI domain or the catalytically inactive inosine-specific nuclease can inhibit or prevent base excision repair of deaminated adenosine residues (e.g., inosine), which can improve the activity or efficiency of the base editor.

[0493] Base editors comprising adenosine deaminase can act on any polynucleotide, including DNA, RNA, and DNA-RNA hybrids. In certain embodiments, base editors comprising adenosine deaminase can deaminate target A of polynucleotides comprising RNA. For example, a base editor may comprise an adenosine deaminase domain, which is capable of deaminating target A of RNA polynucleotides and / or DNA-RNA hybrid polynucleotides. In one embodiment, the adenosine deaminase incorporated into the base editor comprises all or part of an adenosine deaminase acting on RNA (ADAR, e.g., ADAR1 or ADAR2). In another embodiment, the adenosine deaminase incorporated into the base editor comprises all or part of an adenosine deaminase acting on tRNA (ADAT). Base editors comprising adenosine deaminase domains can also deaminate A nucleoside bases of DNA polynucleotides. In one embodiment, the adenosine deaminase of the base editor comprises all or part of ADAT, which comprises one or more mutations that allow the ADAT to deaminate target A in DNA. For example, a base editor may comprise all or part of ADAT from Escherichia coli (EcTadA) comprising one or more of the following mutations: D108N, A106V, D147Y, E155V, L84F, H123Y, I157F, or a corresponding mutation in another adenosine deaminase.

[0494] Adenosine deaminase can be derived from any convenient organism (e.g., E. coli). In some embodiments, the adenosine deaminase is a naturally occurring adenosine deaminase that includes one or more mutations corresponding to any mutation provided in the present disclosure (e.g., mutations in ecTadA). Corresponding residues in any homologous protein can be identified by, for example, sequence alignment and deamination of homologous residues. Mutations in any naturally occurring adenosine deaminase corresponding to any mutation provided in the present disclosure (e.g., any mutation identified in ecTadA) can be generated accordingly.

[0495] In certain embodiments, TadA is any one of the TadAs described in PCT / US2017 / 045381 (WO2018 / 027078), the disclosure of which is incorporated herein by reference in its entirety.

[0496] In certain embodiments, the adenosine deaminase comprises the amino acid sequence:

[0497] Named "TadA reference sequence".

[0498] In some embodiments, the TadA deaminase is a full-length E. coli TadA deaminase. For example, in some embodiments, the adenosine deaminase comprises the amino acid sequence:

[0499]

[0500] However, it should be understood that additional adenosine deaminases useful in the present application will be readily apparent to those skilled in the art and fall within the scope of the present disclosure. For example, the adenosine deaminase can be an adenosine deaminase homolog acting on tRNA (ADAT). Without limitation, the amino acid sequence of an AD AT homolog embodiment includes the following:

[0501] Staphylococcus aureus TadA:

[0502]

[0503] Bacillus subtilis TadA:

[0504]

[0505] Salmonella typhimurium (S.typhimurium) TadA:

[0506]

[0507] S. putrefaciens TadA:

[0508]

[0509] Haemophilus influenzae F3031 (H.influenzae) TadA:

[0510]

[0511] C. crescentus TadA:

[0512]

[0513] Geobacter sulfurreducens (G.sulfurreducens) TadA:

[0514]

[0515] TadA 7.10:

[0516]

[0517] In some embodiments, the adenosine deaminase is obtained from a prokaryotic organism. In some embodiments, the adenosine deaminase is obtained from a bacterium. In some embodiments, the adenosine deaminase is obtained from Escherichia coli, Staphylococcus aureus, Salmonella typhimurium, Shewanella putrefaciens, Haemophilus influenzae, Caulobacter crescentus, or Bacillus subtilis. In some embodiments, the adenosine deaminase is obtained from Escherichia coli.

[0518] In one embodiment, the fusion protein of the invention comprises wild-type TadA linked to TadA 7.10, which is linked to the Cas9 nickase. In a specific embodiment, the fusion protein comprises a single TadA 7.10 domain (e.g., provided as a monomer). In other embodiments, the ABE7.10 editor comprises TadA 7.10 and TadA (wt), which can generate a heterodimer.

[0519] In some embodiments, the adenosine deaminase comprises an amino acid sequence that is at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or at least 99.5 identical to any of the amino acid sequences listed in any adenosine deaminase provided in the disclosure. It is understood that the adenosine deaminase provided in the disclosure may include one or more mutations (e.g., any of the mutations provided in the disclosure). Provided herein are any deaminase domains having a certain percentage of identity plus any of the mutations or a combination described herein. In some embodiments, the adenosine deaminase comprises an amino acid sequence having 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 21, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, or more mutations compared to a reference sequence or any adenosine deaminase provided in the disclosure. In some embodiments, the adenosine deaminase comprises an amino acid sequence having at least 5, at least 10, at least 15, at least 20, at least 25, at least 30, at least 35, at least 40, at least 45, at least 50, at least 60, at least 70, at least 80, at least 90, at least 100, at least 110, at least 120, at least 130, at least 140, at least 150, at least 160, or at least 170 identical contiguous amino acid residues compared to any of the amino acid sequences known in the art or described herein.

[0520] In some embodiments, the adenosine deaminase comprises a D108X mutation, or a corresponding mutation in another adenosine deaminase, relative to the TadA reference sequence, where X indicates any amino acid other than the corresponding amino acid in the wild-type adenosine deaminase. In some embodiments, the adenosine deaminase comprises a D108N, D108V, D108A, or D108Y mutation in the TadA reference sequence, or a corresponding mutation in another adenosine deaminase. It is understood, however, that additional deaminases can be similarly aligned to identify homologous amino acid residues that can be mutated as provided in the present disclosure.

[0521] In some embodiments, the adenosine deaminase comprises an A106X mutation in a TadA reference sequence, or a corresponding mutation in another adenosine deaminase, where X indicates any amino acid other than the corresponding amino acid in the wild-type adenosine deaminase. In some embodiments, the adenosine deaminase comprises an A106V mutation in a TadA reference sequence, or a corresponding mutation in another adenosine deaminase.

[0522] In some embodiments, the adenosine deaminase comprises an E155X mutation in a TadA reference sequence, or a corresponding mutation in another adenosine deaminase, where X indicates any amino acid other than the corresponding amino acid in a wild-type adenosine deaminase. In some embodiments, the adenosine deaminase comprises an E155D, E155G, or E155V mutation in a TadA reference sequence, or a corresponding mutation in another adenosine deaminase.

[0523] In some embodiments, the adenosine deaminase comprises a D147X mutation in a TadA reference sequence, or a corresponding mutation in another adenosine deaminase, where X indicates any amino acid other than the corresponding amino acid in the wild-type adenosine deaminase. In some embodiments, the adenosine deaminase comprises a D147Y mutation in a TadA reference sequence, or a corresponding mutation in another adenosine deaminase.

[0524] It is to be understood that any mutation provided in the present disclosure (e.g., based on the TadA reference sequence amino acid sequence) can be introduced into other adenosine deaminases, such as Staphylococcus aureus TadA (saTadA) or other adenosine deaminases (e.g., bacterial adenosine deaminases). Any mutation identified in the TadA reference sequence can be made in other adenosine deaminases having homologous amino acid residues. It is also to be understood that any mutation provided in the present disclosure can be made in the TadA reference sequence or other adenosine deaminases individually or in any combination.

[0525] For example, relative to the TadA reference sequence, the adenosine deaminase comprises D108N, A106V, E155V, and / or D147Y mutations, or corresponding mutations in another adenosine deaminase. In some embodiments, the adenosine deaminase comprises the following groups of mutations (groups of mutations are separated by ";"), or corresponding mutations in another adenosine deaminase, relative to the TadA reference sequence: D108N and A106V; D108N and E155V; D108N and D147Y; A106V and E155V; A106V and D147Y; E155V and D147Y; D108N, A106V and E55V; D108N, A106V and D147Y; D108N, E55V and D147Y; A106V, E55V and D147Y; and D108N, A106V, E55V and D147Y. However, it should be understood that any combination of corresponding mutations provided in the disclosure can be made in an adenosine deaminase (e.g., ecTadA).

[0526] In some embodiments, the adenosine deaminase comprises one or more of H8X, T17X, ​​L18X, W23X, L34X, W45X, R51X, A56X, E59X, E85X, M94X, I95X, V102X, F104X, A106X, R107X, D108X, K110X, M118X, N127X, A138X, F149X, M151X, R153X, Q154X, I156X, and / or K157X mutations relative to a TadA reference sequence, or one or more corresponding mutations in another adenosine deaminase, wherein the presence of X indicates any amino acid other than the corresponding amino acid in the wild-type adenosine deaminase. In some embodiments, the adenosine deaminase comprises one or more of H8Y, T17S, L18E, W23L, L34S, W45L, R51H, A56E, or A56S, E59G, E85K, or E85G, M94L, 1951, V102A, F104L, A106V, R107C, or R107H, or R107P, D108G, or D108N, or D108V, or D108A, or D108Y, K110I, M118K, N127S, A138V, F149Y, M151V, R153C, Q154L, I156D, and / or K157R mutations relative to a TadA reference sequence, or one or more corresponding mutations in another adenosine deaminase. In some embodiments, the adenosine deaminase comprises one or more of H8X, D108X, and / or N127X mutations relative to a TadA reference sequence, or one or more corresponding mutations in another adenosine deaminase, wherein the presence of X indicates any amino acid other than the corresponding amino acid in the wild-type adenosine deaminase. In some embodiments, the adenosine deaminase comprises one or more of H8Y, D108N, and / or N127S mutations relative to a TadA reference sequence, or one or more corresponding mutations in another adenosine deaminase.

[0527] In some embodiments, the adenosine deaminase comprises one or more of H8X, R26X, M61X, L68X, M70X, A106X, D108X, A109X, N127X, D147X, R152X, Q154X, E155X, K161X, Q163X, and / or T166X mutations relative to a TadA reference sequence, or one or more corresponding mutations in another adenosine deaminase, wherein the presence of X indicates any amino acid other than the corresponding amino acid in the wild-type adenosine deaminase. In some embodiments, the adenosine deaminase comprises one or more of H8Y, R26W, M61I, L68Q, M70V, A106T, D108N, A109T, N127S, D147Y, R152C, Q154H or Q154R, E155G or E155V or E155D, K161Q, Q163H, and / or T166P mutations relative to the TadA reference sequence, or one or more corresponding mutations in another adenosine deaminase.

[0528] In some embodiments, the adenosine deaminase comprises 1, 2, 3, 4, 5, or 6 mutations selected from the group consisting of H8X, D108X, N127X, D147X, R152X, and Q154X, or corresponding mutations in another adenosine deaminase relative to a TadA reference sequence, wherein X indicates the presence of any amino acid other than the corresponding amino acid in the wild-type adenosine deaminase. In some embodiments, the adenosine deaminase comprises 1, 2, 3, 4, 5, 6, 7, or 8 mutations selected from the group consisting of H8X, M61X, M70X, D108X, N127X, Q154X, E155X, and Q163X, or corresponding mutations in another adenosine deaminase relative to a TadA reference sequence, wherein X indicates the presence of any amino acid other than the corresponding amino acid in the wild-type adenosine deaminase. In some embodiments, the adenosine deaminase comprises 1, 2, 3, 4, or 5 mutations selected from the group consisting of H8X, D108X, N127X, E155X, and T166X, or corresponding mutations in another adenosine deaminase relative to a TadA reference sequence, wherein X indicates the presence of any amino acid other than the corresponding amino acid in the wild-type adenosine deaminase.

[0529] In some embodiments, the adenosine deaminase comprises 1, 2, 3, 4, 5, or 6 mutations selected from the group consisting of H8X, A106X, D108X, or corresponding mutations in another adenosine deaminase relative to a TadA reference sequence, wherein X indicates the presence of any amino acid other than the corresponding amino acid in the wild-type adenosine deaminase. In some embodiments, the adenosine deaminase comprises 1, 2, 3, 4, 5, 6, 7, or 8 mutations selected from the group consisting of H8X, R126X, L68X, D108X, N127X, D147X, and E155X, or corresponding mutations in another adenosine deaminase relative to a TadA reference sequence, wherein X indicates the presence of any amino acid other than the corresponding amino acid in the wild-type adenosine deaminase. In some embodiments, the adenosine deaminase comprises 1, 2, 3, 4, or 5 mutations selected from the group consisting of H8X, D108X, A109X, N127X, and E155X, or corresponding mutations in another adenosine deaminase relative to a TadA reference sequence, wherein X indicates the presence of any amino acid other than the corresponding amino acid in the wild-type adenosine deaminase.

[0530] In some embodiments, the adenosine deaminase comprises 1, 2, 3, 4, 5, or 6 mutations selected from the group consisting of H8Y, D108N, N127S, D147Y, R152C, and Q154H relative to the TadA reference sequence, or corresponding mutations in another adenosine deaminase. In some embodiments, the adenosine deaminase comprises 1, 2, 3, 4, 5, 6, 7, or 8 mutations selected from the group consisting of H8Y, M61I, M70V, D108N, N127S, Q154R, E155G, and Q163H relative to the TadA reference sequence, or corresponding mutations in another adenosine deaminase. In some embodiments, the adenosine deaminase comprises 1, 2, 3, 4, or 5 mutations selected from the group consisting of H8Y, D108N, N127S, E155V, and T166P relative to the TadA reference sequence, or corresponding mutations in another adenosine deaminase. In some embodiments, the adenosine deaminase comprises 1, 2, 3, 4, 5, or 6 mutations selected from the group consisting of H8Y, A106T, D108N, N127S, E155D, and K161Q relative to the TadA reference sequence, or corresponding mutations in another adenosine deaminase. In some embodiments, the adenosine deaminase comprises 1, 2, 3, 4, 5, 6, 7, or 8 mutations selected from the group consisting of H8Y, R126W, L68Q, D108N, N127S, D147Y, and E155V, or corresponding mutations in another adenosine deaminase, relative to a TadA reference sequence. In some embodiments, the adenosine deaminase comprises 1, 2, 3, 4, or 5 mutations selected from the group consisting of H8Y, D108N, A109T, N127S, and E155G, or corresponding mutations in another adenosine deaminase, relative to a TadA reference sequence.

[0531] It is understood that any mutation provided in the present disclosure and any additional mutations (e.g., based on the TadA reference sequence amino acid sequence) can be introduced into other adenosine deaminases. Any mutation provided in the present disclosure can be made individually or in any combination in the TadA reference sequence or other adenosine deaminases.

[0532] Details of the A to G nucleoside base editing protein are described in International PCT Application Nos. PCT / 2017 / 045381 (WO2018 / 027078) and Gaudelli, NM, et al., "Programmable base editing of A·T to G·C in genomic DNA without DNA cleavage", Nature 551, 464-471 (2017), all of which are incorporated by reference into the disclosure of this specification.

[0533] In some embodiments, the adenosine deaminase comprises one or more of the mutations in the TadA reference sequence, or one or more corresponding mutations in another adenosine deaminase. In some embodiments, the adenosine deaminase comprises the A106V and D108N mutations in the TadA reference sequence, or corresponding mutations in another adenosine deaminase. In some embodiments, the adenosine deaminase comprises the R107C and D108N mutations in the TadA reference sequence, or corresponding mutations in another adenosine deaminase. In some embodiments, the adenosine deaminase comprises the H8Y, D108N, N127S, D147Y, and Q154H mutations in the TadA reference sequence, or corresponding mutations in another adenosine deaminase. In some embodiments, the adenosine deaminase comprises the H8Y, R24W, D108N, N127S, D147Y, and E155V mutations in the TadA reference sequence, or corresponding mutations in another adenosine deaminase. In some embodiments, the adenosine deaminase comprises the D108N, D147Y, and E155V mutations in the TadA reference sequence, or the corresponding mutations in another adenosine deaminase. In some embodiments, the adenosine deaminase comprises the H8Y, D108N, and N127S mutations in the TadA reference sequence, or the corresponding mutations in another adenosine deaminase. In some embodiments, the adenosine deaminase comprises the A106V, D108N, D147Y, and E155V mutations in the TadA reference sequence, or the corresponding mutations in another adenosine deaminase.

[0534] In some embodiments, the adenosine deaminase comprises one or more of a, S2X, H8X, I49X, L84X, H123X, N127X, I156X, and / or K160X mutations in the TadA reference sequence, or corresponding mutations in another adenosine deaminase, wherein the presence of X indicates any amino acid other than the corresponding amino acid in the wild-type adenosine deaminase. In some embodiments, the adenosine deaminase comprises one or more of S2A, H8Y, I49F, L84F, H123Y, N127S, I156F, and / or K160S mutations in the TadA reference sequence, or corresponding mutations in another adenosine deaminase.

[0535] In some embodiments, the adenosine deaminase comprises an L84X mutant adenosine deaminase, where X indicates any amino acid other than the corresponding amino acid in the wild-type adenosine deaminase. In some embodiments, the adenosine deaminase comprises an L84F mutation in a TadA reference sequence, or a corresponding mutation in another adenosine deaminase.

[0536] In some embodiments, the adenosine deaminase comprises an H123X mutation in a TadA reference sequence, or a corresponding mutation in another adenosine deaminase, wherein X indicates any amino acid other than the corresponding amino acid in a wild-type adenosine deaminase. In some embodiments, the adenosine deaminase comprises an H123Y mutation in a TadA reference sequence, or a corresponding mutation in another adenosine deaminase. In some embodiments, the adenosine deaminase comprises an I157X mutation in a TadA reference sequence, or a corresponding mutation in another adenosine deaminase, wherein X indicates any amino acid other than the corresponding amino acid in a wild-type adenosine deaminase. In some embodiments, the adenosine deaminase comprises an I157F mutation in a TadA reference sequence, or a corresponding mutation in another adenosine deaminase.

[0537] In some embodiments, the adenosine deaminase comprises 1, 2, 3, 4, 5, 6, or 7 mutations in a TadA reference sequence selected from the group consisting of L84X, A106X, D108X, H123X, D147X, E155X, and I156X, or corresponding mutations in another adenosine deaminase, wherein X indicates the presence of any amino acid other than the corresponding amino acid in the wild-type adenosine deaminase. In some embodiments, the adenosine deaminase comprises 1, 2, 3, 4, 5, or 6 mutations in a TadA reference sequence selected from the group consisting of S2X, I49X, A106X, D108X, D147X, and E155X, or corresponding mutations in another adenosine deaminase, wherein X indicates the presence of any amino acid other than the corresponding amino acid in the wild-type adenosine deaminase. In some embodiments, the adenosine deaminase comprises 1, 2, 3, 4, or 5 mutations in a TadA reference sequence selected from the group consisting of H8X, A106X, D108X, N127X, and K160X, or corresponding mutations in another adenosine deaminase, wherein X indicates the presence of any amino acid other than the corresponding amino acid in the wild-type adenosine deaminase.

[0538] In some embodiments, the adenosine deaminase comprises 1, 2, 3, 4, 5, 6, or 7 mutations in the TadA reference sequence selected from the group consisting of L84F, A106V, D108N, H123Y, D147Y, E155V, and I156F, or corresponding mutations in another adenosine deaminase. In some embodiments, the adenosine deaminase comprises 1, 2, 3, 4, 5, or 6 mutations in the TadA reference sequence selected from the group consisting of S2A, I49F, A106V, D108N, D147Y, and E155V, or corresponding mutations in another adenosine deaminase.

[0539] In some embodiments, the adenosine deaminase comprises 1, 2, 3, 4, or 5 mutations in a TadA reference sequence selected from the group consisting of H8Y, A106T, D108N, N127S, and K160S, or corresponding mutations in another adenosine deaminase.

[0540] In some embodiments, the adenosine deaminase comprises one or more of the E25X, R26X, R107X, A142X, and / or A143X mutations in the TadA reference sequence, or one or more corresponding mutations in another adenosine deaminase, wherein the presence of X indicates any amino acid other than the corresponding amino acid in the wild-type adenosine deaminase. In some embodiments, the adenosine deaminase comprises one or more of the E25M, E25D, E25A, E25R, E25V, E25S, E25Y, R26G, R26N, R26Q, R26C, R26L, R26K, R107P, R07K, R107A, R107N, R107W, R107H, R107S, A142N, A142D, A142G, A143D, A143G, A143E, A143L, A143W, A143M, A143S, A143Q, and / or A143R mutations in the TadA reference sequence, or one or more corresponding mutations in another adenosine deaminase. In several embodiments, the adenosine deaminase comprises one or more of the mutations described herein corresponding to the TadA reference sequence, or one or more corresponding mutations in another adenosine deaminase.

[0541] In some embodiments, the adenosine deaminase comprises an E25X mutation in a TadA reference sequence, or a corresponding mutation in another adenosine deaminase, where X indicates any amino acid other than the corresponding amino acid in the wild-type adenosine deaminase. In some embodiments, the adenosine deaminase comprises an E25M, E25D, E25A, E25R, E25V, E25S, or E25Y mutation in a TadA reference sequence, or a corresponding mutation in another adenosine deaminase.

[0542] In some embodiments, the adenosine deaminase comprises an R26X mutation in a TadA reference sequence, or a corresponding mutation in another adenosine deaminase, where X indicates any amino acid other than the corresponding amino acid in the wild-type adenosine deaminase. In some embodiments, the adenosine deaminase comprises an R26G, R26N, R26Q, R26C, R26L, or R26K mutation in a TadA reference sequence, or a corresponding mutation in another adenosine deaminase.

[0543] In some embodiments, the adenosine deaminase comprises a R107X mutation in a TadA reference sequence, or a corresponding mutation in another adenosine deaminase, where X indicates any amino acid other than the corresponding amino acid in the wild-type adenosine deaminase. In some embodiments, the adenosine deaminase comprises a R107P, R07K, R107A, R107N, R107W, R107H, or R107S mutation in a TadA reference sequence, or a corresponding mutation in another adenosine deaminase.

[0544] In some embodiments, the adenosine deaminase comprises an A142X mutation in a TadA reference sequence, or a corresponding mutation in another adenosine deaminase, where X indicates any amino acid other than the corresponding amino acid in a wild-type adenosine deaminase. In some embodiments, the adenosine deaminase comprises an A142N, A142D, A142G mutation in a TadA reference sequence, or a corresponding mutation in another adenosine deaminase.

[0545] In some embodiments, the adenosine deaminase comprises an A143X mutation in a TadA reference sequence, or a corresponding mutation in another adenosine deaminase, where X indicates any amino acid other than the corresponding amino acid in a wild-type adenosine deaminase. In some embodiments, the adenosine deaminase comprises an A143D, A143G, A143E, A143L, A143W, A143M, A143S, A143Q and / or A143R mutation in a TadA reference sequence, or a corresponding mutation in another adenosine deaminase.

[0546] In some embodiments, the adenosine deaminase comprises one or more of the H36X, N37X, P48X, I49X, R51X, M70X, N72X, D77X, E134X, S146X, Q154X, K157X, and / or K161X mutations in the TadA reference sequence, or one or more corresponding mutations in another adenosine deaminase, wherein the presence of X indicates any amino acid other than the corresponding amino acid in the wild-type adenosine deaminase. In some embodiments, the adenosine deaminase comprises one or more of the H36L, N37T, N37S, P48T, P48L, I49V, R51H, R51L, M70L, N72S, D77G, E134G, S146R, S146C, Q154H, K157N, and / or K161T mutations in the TadA reference sequence, or one or more corresponding mutations in another adenosine deaminase.

[0547] In some embodiments, the adenosine deaminase comprises an H36X mutation in a TadA reference sequence, or a corresponding mutation in another adenosine deaminase, where X indicates any amino acid other than the corresponding amino acid in the wild-type adenosine deaminase. In some embodiments, the adenosine deaminase comprises an H36L mutation in a TadA reference sequence, or a corresponding mutation in another adenosine deaminase.

[0548] In some embodiments, the adenosine deaminase comprises an N37X mutation in a TadA reference sequence, or a corresponding mutation in another adenosine deaminase, where X indicates any amino acid other than the corresponding amino acid in the wild-type adenosine deaminase. In some embodiments, the adenosine deaminase comprises an N37T, or N37S mutation in a TadA reference sequence, or a corresponding mutation in another adenosine deaminase.

[0549] In some embodiments, the adenosine deaminase comprises a P48X mutation in a TadA reference sequence, or a corresponding mutation in another adenosine deaminase, where X indicates any amino acid other than the corresponding amino acid in the wild-type adenosine deaminase. In some embodiments, the adenosine deaminase comprises a P48T, or P48L mutation in a TadA reference sequence, or a corresponding mutation in another adenosine deaminase.

[0550] In some embodiments, the adenosine deaminase comprises an R51X mutation in a TadA reference sequence, or a corresponding mutation in another adenosine deaminase, wherein X indicates any amino acid other than the corresponding amino acid in the wild-type adenosine deaminase. In some embodiments, the adenosine deaminase comprises an R51H, or R51L mutation in a TadA reference sequence, or a corresponding mutation in another adenosine deaminase.

[0551] In some embodiments, the adenosine deaminase comprises an S146X mutation in a TadA reference sequence, or a corresponding mutation in another adenosine deaminase, where X indicates any amino acid other than the corresponding amino acid in a wild-type adenosine deaminase. In some embodiments, the adenosine deaminase comprises an S146R, or S146C mutation in a TadA reference sequence, or a corresponding mutation in another adenosine deaminase.

[0552] In some embodiments, the adenosine deaminase comprises a K157X mutation in a TadA reference sequence, or a corresponding mutation in another adenosine deaminase, where X indicates any amino acid other than the corresponding amino acid in the wild-type adenosine deaminase. In some embodiments, the adenosine deaminase comprises a K157N mutation in a TadA reference sequence, or a corresponding mutation in another adenosine deaminase.

[0553] In some embodiments, the adenosine deaminase comprises a P48X mutation in a TadA reference sequence, or a corresponding mutation in another adenosine deaminase, where X indicates any amino acid other than the corresponding amino acid in the wild-type adenosine deaminase. In some embodiments, the adenosine deaminase comprises a P48S, P48T, or P48A mutation in a TadA reference sequence, or a corresponding mutation in another adenosine deaminase.

[0554] In some embodiments, the adenosine deaminase comprises an A142X mutation in a TadA reference sequence, or a corresponding mutation in another adenosine deaminase, where X indicates any amino acid other than the corresponding amino acid in the wild-type adenosine deaminase. In some embodiments, the adenosine deaminase comprises an A142N mutation in a TadA reference sequence, or a corresponding mutation in another adenosine deaminase.

[0555] In some embodiments, the adenosine deaminase comprises a W23X mutation in a TadA reference sequence, or a corresponding mutation in another adenosine deaminase, where X indicates any amino acid other than the corresponding amino acid in the wild-type adenosine deaminase. In some embodiments, the adenosine deaminase comprises a W23R, or W23L mutation in a TadA reference sequence, or a corresponding mutation in another adenosine deaminase.

[0556] In some embodiments, the adenosine deaminase comprises an R152X mutation in a TadA reference sequence, or a corresponding mutation in another adenosine deaminase, wherein X indicates any amino acid other than the corresponding amino acid in a wild-type adenosine deaminase. In some embodiments, the adenosine deaminase comprises an R152P, or R152H mutation in a TadA reference sequence, or a corresponding mutation in another adenosine deaminase.

[0557] In one embodiment, the adenosine deaminase may comprise mutations H36L, R51L, L84F, A106V, D108N, H123Y, S146C, D147Y, E155V, I156F, and K157N. In some embodiments, relative to the TadA reference sequence, the adenosine deaminase comprises a combination of the following mutations, wherein each mutation in the combination is separated by a "-" and each mutation combination is enclosed in brackets: (A106V_D108N), (R107C_D108N),

[0558] (H8Y_D108N_N127S_D 147Y_Q154H),(H8Y_R24W_D108N_N127S_D147Y_E155V),(D108N_D147Y_E155V),(H8Y_D108N_N127S),(H8Y_D108N_N127S_D147Y_Q154H),(A106V_D108N_D147Y_E155V)(D108Q_D147Y_E155V)(D108M_D147Y_E155V),(D108L_D147Y_E155V),(D108K_D147Y_E155V),(D108I_D147Y_E155V),

[0559] (D108F_D147Y_E155V),(A106V_D108N_D147Y),(A106V_D108M_D147Y_E155V),(E59A_A106V_D108N_D147Y_E155V),(E59A cat dead_A106V_D108N_D147Y_E155V),(L84F_A106V_D108N_H123Y_D147Y_E155V_I156Y),

[0560] (L84F_A106V_D108N_H123Y_D147Y_E155V_I156F),(D103A_D104N),(G22P_D103A_D104N),(G22P_D103A_D104N_S138A),(D103A_D104N_S138A),(R26G_L84F_A106V_R107H_D108N_H123Y_A142N_A143D_D147Y_E155V_I156F),(E25G_R26G_L84F_A106V_R107H_D108N_H123Y_A142N_A143D_D147Y_E155V_I156F),

[0561] (E25D_R26G_L84F_A106V_R107K_D108N_H123Y_A142N_A143G_D147Y_E155V_I156F),(R26Q_L84F_A106V_D108N_H123Y_A142N_D147Y_E155V_1156F),(E25M_R26G_L84F_A106V_R107P_D108N_H123Y_A142N_A143D_D147Y_E155V_I156F),(R26C_L84F_A106V_R107H_D108N_H123Y_A142N_D147Y_E155V_I156F),(L84F_A106V_D108N_H123Y_A142N_A143L_D147Y_E155V_I156F),(R26G_L84F_A106V_D108N_H123Y_A142N_D147Y_E155V_I156F),

[0562] (E25A_R26G_L84F_A106V_R107N_D108N_H123Y_A142N_A143E_D147Y_E155V_1156F),

[0563] (R26G_L84F_A106V_R107H_D108N_H123Y_A142N_A143D_D147Y_E155V_I156F),

[0564] (A106V_D108N_A142N_D147Y_E155V),

[0565] (R26G_A106V_D108N_A142N_D147Y_E155V),

[0566] (E25D_R26G_A106V_R107K_D108N_A142N_A143G_D147Y_E155V),

[0567] (R26G_A106V_D108N_R107H_A142N_A143D_D147Y_E155V),

[0568] (E25D_R26G_A106V_D108N_A142N_D147Y_E155V),

[0569] (A106V_R107K_D108N_A142N_D147Y_E155V),

[0570] (A106V_D108N_A142N_A143G_D147Y_E155V),

[0571] (A106V_D108N_A142N_A143L_D147Y_E155V),

[0572] (H36L_R51L_L84F_A106V_D108N_H123Y_S146C_D147Y_E155V_I156F_K157N),

[0573] (N37T_P48T_M70L_L84F_A106V_D108N_H123Y_D147Y_I49V_E155V_I156F),

[0574] (N37S_L84F_A106V_D108N_H123Y_D147Y_E155V_I156F_K161T),

[0575] (H36L_L84F_A106V_D108N_H123Y_D147Y_Q154H_E155V_I156F),

[0576] (N72S_L84F_A106V_D108N_H123Y_S146R_D147Y_E155V_I156F),

[0577] (H36L_P48L_L84F_A106V_D108N_H123Y_E134G_D147Y_E155V_I156F),

[0578] (H36L_L84F_A106V_D108N_H123Y_D147Y_E155V_I156F_K157N),

[0579] (H36L_L84F_A106V_D108N_H123Y_S146C_D147Y_E155V_I156F),

[0580] (L84F_A106V_D108N_H123Y_S146R_D147Y_E155V_I156F_K161T),

[0581] (N37S_R51H_D77G_L84F_A106V_D108N_H123Y_D147Y_E155V_I156F),

[0582] (R51L_L84F_A106V_D108N_H123Y_D147Y_E155V_I156F_K157N),

[0583] (D24G_Q71R_L84F_H96L_A106V_D108N_H123Y_D147Y_E155V_1156F_K160E),

[0584] (H36L_G67V_L84F_A106V_D108N_H123Y_S146T_D147Y_E155V_I156F),

[0585] (Q71L_L84F_A106V_D108N_H123Y_L137M_A143E_D147Y_E155V_I156F),

[0586] (E25G_L84F_A106V_D108N_H123Y_D147Y_E155V_I156F_Q159L),

[0587] (L84F_A91T_F104I_A106V_D108N_H123Y_D147Y_E155V_I156F),

[0588] (N72D_L84F_A106V_D108N_H123Y_G125A_D147Y_E155V_I156F),

[0589] (P48S_L84F_S97C_A106V_D108N_H123Y_D147Y_E155V_I156F),

[0590] (W23G_L84F_A106V_D108N_H123Y_D147Y_E155V_I156F),

[0591] (D24G_P48L_Q71R_L84F_A106V_D108N_H123Y_D147Y_E155V_I156F_Q159L),

[0592] (L84F_A106V_D108N_H123Y_A142N_D147Y_E155V_I156F),

[0593] (H36L_R51L_L84F_A106V_D108N_H123Y_A142N_S146C_D147Y_E155V_I156F_K157N),(N37S_L84F_A106V_D108N_H123Y_A142N_D147Y_E155V_I156F_K161T),(L84F_A106V_D108N_D147Y_E155V_I156F),

[0594] (R51L_L84F_A106V_D108N_H123Y_S146C_D147Y_E155V_I156F_K157N_K161T),

[0595] (L84F_A106V_D108N_H123Y_S146C_D147Y_E155V_I156F_K161T),

[0596] (L84F_A106V_D108N_H123Y_S146C_D147Y_E155V_I156F_K157N_K160E_K161T),

[0597] (L84F_A106V_D108N_H123Y_S146C_D147Y_E155V_I156F_K157N_K160E),(R74QL84F_A106V_D108N_H123Y_D147Y_E155V_I156F),

[0598] (R74A_L84F_A106V_D108N_H123Y_D147Y_E155V_I156F),

[0599] (L84F_A106V_D108N_H123Y_D147Y_E155V_I156F),

[0600] (R74Q_L84F_A106V_D108N_H123Y_D147Y_E155V_I156F),

[0601] (L84F_R98Q_A106V_D108N_H123Y_D147Y_E155V_I156F),

[0602] (L84F_A106V_D108N_H123Y_R129Q_D147Y_E155V_I156F),

[0603] (P48S_L84F_A106V_D108N_H123Y_A142N_D147Y_E155V_I156F),(P48S_A142N),

[0604] (P48T_I49V_L84F_A106V_D108N_H123Y_A142N_D147Y_E155V_I156F_L157N),(P48T_I49V_A142N),

[0605] (H36L_P48S_R51L_L84F_A106V_D108N_H123Y_S146C_D147Y_E155V_I156F_K157N),(H36L_P48S_R51L_L84F_A106V_D108N_H123Y_S146C_A142N_D147Y_E155V_I156F(H36L_P48T_I49V_R51L_L84F_A106V_D108N_H123Y_S146C_D147Y_E155V_1156FK157N),

[0606] (H36L_P48T_I49V_R51L_L84F_A106V_D108N_H123Y_A142N_S146C_D147Y_E155V_I156F_K157N),

[0607] (H36L_P48A_R5IL_L84F_A106V_D108N_H123Y_S146C_D147Y_E155V_I156F_K157N),(H36L_P48A_R5IL_L84F_A106V_D108N_H123Y_A142N_S146C_D147Y_E155V_I156FK157N),

[0608] (H36L_P48A_R51L_L84F_A106V_D108N_H123Y_S146C_A142N_D147Y_E155V_I156F_K157N),

[0609] (W23L_H36L_P48A_R51L_L84F_A106V_D108N_H123Y_S146C_D147Y_E155V_I156F_K157N),

[0610] (W23R_H36L_P48A_R51L_L84F_A106V_D108N_H123Y_S146C_D147Y_E155V_I156F_K157N),

[0611] (W23L_H36L_P48A_R51L_L84F_A106V_D108N_H123Y_S146R_D147Y_E155V_I156F_K161T),

[0612] (H36L_P48A_R51L_L84F_A106V_D108N_H123Y_S146C_D147Y_R152H_E155V_I156F_K157N),

[0613] (H36L_P48A_R51L_L84F_A106V_D108N_H123Y_S146C_D147Y_R152P_E155V_I156F_K157N),

[0614] (W23L_H36L_P48A_R51L_L84F_A106V_D108N_H123Y_S146C_D147Y_R152P_E155V_I156F_K157N),

[0615] (W23L_H36L_P48A_R51L_L84F_A106V_D108N_H123Y_A142A_S146C_D147Y_E155V_I156F_K157N),

[0616] (W23L_H36L_P48A_R51L_L84F_A106V_D108N_H123Y_A142A_S146C_D147Y_R152P_E155V_I156F_K157N),

[0617] (W23L_H36L_P48A_R51L_L84F_A106V_D108N_H123Y_S146R_D147Y_E155V_I156F_K161T),

[0618] (W23R_H36L_P48A_R51L_L84F_A106V_D108N_H123Y_S146C_D147Y_R152P_E155V_I156F_K157N),

[0619] (H36L_P48A_R51L_L84F_A106V_D108N_H123Y_A142N_S146C_D147Y_R152P_E155V_I156F_K157N).

[0620] In certain embodiments, the fusion proteins provided in the present disclosure include one or more features that improve the base editing activity of the fusion protein. For example, any of the fusion proteins provided in the present disclosure may include a Cas9 domain with reduced nuclease activity. In several embodiments, any of the fusion proteins provided in the present disclosure may have a Cas9 domain that does not have nuclease activity; or a Cas9 domain that cuts one strand of a diploid DNA molecule, referred to as a Cas9 nickase (nCas9).

[0621] Cytidine deaminase

[0622] In one embodiment, the fusion protein of the present invention comprises a cytidine deaminase. In several embodiments, the cytidine deaminase provided in the present disclosure can deaminate cytosine or 5-methylcytosine to uracil or thymine. In one embodiment, the cytidine deaminase provided in the present disclosure can deaminate cytosine in DNA. The cytidine deaminase can be derived from any suitable organism. In several embodiments, the cytidine deaminase is a naturally occurring cytidine deaminase, which includes one or more mutations corresponding to any mutation provided in the present disclosure. A person skilled in the art will be able to identify the corresponding residues in any homologous protein, for example, by sequence alignment and deamination of homologous residues. Accordingly, a person skilled in the art will be able to generate mutations corresponding to any mutation provided in the present disclosure in any naturally occurring cytidine deaminase. In several embodiments, the cytidine deaminase is obtained from a prokaryotic organism. In several embodiments, the cytidine deaminase is obtained from bacteria. In several embodiments, the cytidine deaminase is obtained from mammals (e.g., humans).

[0623] In some embodiments, the cytidine deaminase comprises an amino acid sequence that is at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or at least 99.5% identical to any of the cytidine deaminase amino acid sequences listed in the disclosure. It is understood that the cytidine deaminase provided in the disclosure may include one or more mutations (e.g., any of the mutations provided in the disclosure). Provided herein are any deaminase domains having a certain percentage of identity plus any of the mutations described herein or a combination thereof. In some embodiments, the cytidine deaminase comprises an amino acid sequence having 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 21, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, or more mutations compared to a reference sequence or any cytidine deaminase provided in the disclosure. In some embodiments, the cytidine deaminase comprises an amino acid sequence having at least 5, at least 10, at least 15, at least 20, at least 25, at least 30, at least 35, at least 40, at least 45, at least 50, at least 60, at least 70, at least 80, at least 90, at least 100, at least 110, at least 120, at least 130, at least 140, at least 150, at least 160, or at least 170 identical contiguous amino acid residues compared to any of the amino acid sequences known in the art or described herein.

[0624] The fusion protein of the present invention comprises a nucleic acid editing domain. In some embodiments, the nucleic acid editing domain can catalyze a C to U base change. In some embodiments, the nucleic acid editing domain is a deaminase domain. In some embodiments, the deaminase is a cytidine deaminase or an adenosine deaminase. In some embodiments, the deaminase is an apolipoprotein B mRNA editing complex (APOBEC) family deaminase. In some embodiments, the deaminase is an APOBEC1 deaminase. In some embodiments, the deaminase is an APOBEC2 deaminase. In some embodiments, the deaminase is an APOBEC3 deaminase. In some embodiments, the deaminase is an APOBEC3A deaminase. In some embodiments, the deaminase is an APOBEC3B deaminase. In some embodiments, the deaminase is an APOBEC3C deaminase. In some embodiments, the deaminase is an APOBEC3D deaminase. In some embodiments, the deaminase is an APOBEC3E deaminase. In some embodiments, the deaminase is an APOBEC3F deaminase. In some embodiments, the deaminase is an APOBEC3G deaminase. In some embodiments, the deaminase is an APOBEC3H deaminase. In some embodiments, the deaminase is an APOBEC4 deaminase. In some embodiments, the deaminase is an activation-induced deaminase (AID). In some embodiments, the deaminase is a vertebrate deaminase. In some embodiments, the deaminase is an invertebrate deaminase. In some embodiments, the deaminase is a human, chimpanzee, orangutan, monkey, bovine, canine, rat, or mouse deaminase. In some embodiments, the deaminase is a human deaminase. In some embodiments, the deaminase is a rat deaminase, e.g., rAPOBEC1. In some embodiments, the deaminase is lamprey cytidine deaminase 1 (pmCDA1). In some embodiments, the deaminase is human APOBEC3G. In some embodiments, the deaminase is a fragment of human APOBEC3G. In some embodiments, the deaminase is a human APOBEC3G variant comprising a D316R D317R mutation. In some embodiments, the deaminase is a fragment of human APOBEC3G and its corresponding mutation comprising a D316R D317R mutation. In some embodiments, the nucleic acid editing domain is at least 80%, at least 85%, at least 90%, at least 92%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or at least 99.5% identical to the deaminase domain of any deaminase described herein.

[0625] Cas9 domains of nucleoside base editors

[0626] In some aspects, the nucleic acid programmable DNA binding protein (napDNAbp) is a Cas9 domain. Non-limiting examples of Cas9 domains are provided in this disclosure. The Cas9 domain can be a nuclease-active Cas9 domain, a nuclease-inactive Cas9 domain, or a Cas9 nickase. In some embodiments, the Cas9 domain is a nuclease-active Cas9 domain. For example, the Cas9 domain can be a Cas9 domain that cuts two strands of a diploid nucleic acid (e.g., two strands of a diploid DNA molecule). In some embodiments, the Cas9 domain comprises any one of the amino acid sequences listed in this disclosure. In some embodiments, the Cas9 domain comprises an amino acid sequence that is at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or at least 99.5% identical to any one of the amino acid sequences listed in this disclosure. In some embodiments, the Cas9 domain comprises an amino acid sequence having 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 21, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, or more mutations compared to any of the amino acid sequences listed in the disclosure. In some embodiments, the Cas9 domain comprises an amino acid sequence having at least 10, at least 15, at least 20, at least 30, at least 40, at least 50, at least 60, at least 70, at least 80, at least 90, at least 100, at least 150, at least 200, at least 250, at least 300, at least 350, at least 400, at least 500, at least 600, at least 700, at least 800, at least 900, at least 1000, at least 1100, or at least 1200 identical contiguous amino acid residues compared to any of the amino acid sequences listed in the disclosure.

[0627] In some embodiments, the Cas9 domain is a nuclease-inactivated Cas9 domain (dCas9). For example, the dCas9 domain can bind to a diploid nucleic acid molecule (e.g., via a gRNA molecule) without cleaving any of the strands of the diploid nucleic acid molecule. In some embodiments, the nuclease-inactivated dCas9 domain comprises a D10X mutation and an H840X mutation of an amino acid sequence listed in the disclosure, or a corresponding mutation of any amino acid sequence provided in the disclosure, wherein X is any amino acid change. In some embodiments, the nuclease-inactivated dCas9 domain comprises a D10A mutation and an H840A mutation of an amino acid sequence listed in the disclosure, or a corresponding mutation of any amino acid sequence provided in the disclosure.

[0628] In some embodiments, the Cas9 domain is a Cas9 nickase. The Cas9 nickase can be a Cas9 protein that can cleave only one strand of a diploid nucleic acid molecule (e.g., a diploid DNA molecule). In some embodiments, the Cas9 nickase cleaves the target strand of a diploid nucleic acid molecule, meaning that the Cas9 nickase cleaves the strand that is base paired with (complementary to) a gRNA (e.g., sgRNA) bound to Cas9. In some embodiments, the Cas9 nickase comprises a D10A mutation and has a histidine at position 840. In some embodiments, the Cas9 nickase cleaves a non-target and non-base editing strand of a diploid nucleic acid molecule, meaning that the Cas9 nickase cleaves the strand that is not base paired with a gRNA (e.g., sgRNA) bound to Cas9. In some embodiments, the Cas9 nickase comprises an H840A mutation and has an asparagine, or a corresponding mutation, at position 10. In some embodiments, the Cas9 nickase comprises an amino acid sequence that is at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or at least 99.5% identical to any of the Cas9 nickases provided in the present disclosure. Any suitable Cas9 nickase will be apparent to those of skill in the art based on the disclosure herein and the knowledge in the art and is within the scope of the disclosure herein.

[0629] Cas9 domains with reduced exclusivity

[0630] Typically, Cas9 proteins, such as Cas9 from Streptococcus pyogenes (spCas9), require a canonical NGG PAM sequence to bind to a specific nucleic acid region, where the "N" in "NGG" is adenine (A), thymine (T), or cytosine (C), and G is guanine. This may limit the ability to edit the desired base in the genome. In some embodiments, the base editing fusion proteins provided in the present disclosure may need to be placed in a precise position, such as a region containing the target base upstream of the PAM. See, for example, Komor, AC, et al., "Programmable base editing of target bases in genomic DNA without double-stranded DNA cleavage," Nature 533, 420-424 (2016), which is incorporated by reference into the disclosure of this specification in its entirety. Accordingly, in some embodiments, any of the fusion proteins provided in the present disclosure may contain a Cas9 domain that can bind to a nucleotide sequence that does not contain a canonical (e.g., NGG) PAM sequence. Cas9 domains that bind to non-canonical PAM sequences have been described in the art and are apparent to those skilled in the art. For example, Cas9 domains that bind to non-canonical PAM sequences have been described in Kleinstiver, BP, et al., "Engineered CRISPR-Cas9 with altered PAM specificity" Nature 523, 481-485 (2015); and Kleinstiver, BP, et al., "Expanding the targeting range of Staphylococcus aureus CRISPR-Cas9 by modifying PAM recognition" Nature Biotechnology 33, 1293-1298 (2015); Nishimasu, H., et al., "Engineered CRISPR-Cas9 nuclease with expanded targeting space" Science. 2018 Sep 21; 361(6408): 1259-1262; Chatterjee, P., et al., "Minimal PAM specificity of highly similar SpCas9 homologues" PAM specificity of a highly similar SpCas9 ortholog”) Sci Adv. 2018 Oct 24; 4(10):eaau0766.doi:10.1126 / sciadv.aau0766, each of which is incorporated by reference into the disclosure of this specification. Several PAM variants are described in Table 1 below.

[0631] Table 1. Cas9 proteins and corresponding PAM sequences

[0632] Variants PAM spCas9 NGG spCas9-VRQR NGA spCas9-VRER NGCG xCas9(sp) NGN saCas9 NNGRRT saCas9-KKH NNNRRT spCas9-MQKSER NGCG spCas9-MQKSER NGCN spCas9-LRKIQK NGTN spCas9-LRVSQK NGTN spCas9-LRVSQL NGTN SpyMacCas9 NAA Cpf1 5'(TTTV)

[0633] Cas9 complex with guide RNA

[0634] Some aspects of the present disclosure provide a complex comprising any of the fusion proteins provided in the present disclosure and a guide RNA (e.g., a guide targeting a gene of interest). Any method for linking fusion protein domains may be used (e.g., from the form (GGGS) n 、(GGGGS) n , and (G) n Highly flexible link base to form (EAAAK) n 、(SGGS) n , SGSETPGTSESATPES (for example, refer to Guilinger JP, Thompson DB, Liu DR. "Fusion of catalytically inactive Cas9 toFokI nuclease improves the specificity of genome modification." Nat. Biotechnol. 2014; 32(6): 577-82, the entire disclosure of which is incorporated by reference into this specification) and (XP) n In some embodiments, n is 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15. In some embodiments, the linker comprises (GGS) n motif, wherein n is 1, 3, or 7. In some embodiments, the Cas9 domain of the fusion protein provided in the present disclosure is fused via a linker comprising the amino acid sequence SGSETPGTSESATPES:.

[0635] In some embodiments, the guide nucleic acid (e.g., guide RNA) is 15-100 nucleotides in length and comprises a sequence of at least 10 contiguous nucleotides that is complementary to the target sequence. In some embodiments, the guide RNA is 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, or 50 nucleotides in length. In some embodiments, the guide RNA comprises a sequence of 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, or 40 contiguous nucleotides that are complementary to the target sequence. In some embodiments, the target sequence is a DNA sequence. In some embodiments, the target sequence is a sequence in the genome of bacteria, yeast, fungi, insects, plants, or animals. In some embodiments, the target sequence is a sequence in the genome of a human. In some embodiments, the 3' end of the target sequence is adjacent to a canonical PAM sequence (NGG). In some embodiments, the target sequence is a sequence in the genome of a human. In some embodiments, the 3' end of the target sequence is adjacent to a non-canonical PAM sequence (e.g., a sequence listed in Table 1 or 5'-NAA-3'). In some embodiments, the guide nucleic acid (eg, guide RNA) is complementary to a sequence in a gene of interest.

[0636] Some aspects of the present disclosure provide methods for using fusion proteins or complexes provided in the present disclosure. For example, some aspects of the present disclosure provide methods comprising contacting a DNA molecule with any of the fusion proteins provided in the present disclosure, and contacting with at least one guide RNA, wherein the guide RNA is about 15-100 nucleotides in length and comprises a sequence of at least 10 adjacent nucleotides that are complementary to the target sequence. In some embodiments, the 3' end of the target sequence is adjacent to the AGC, GAG, TTT, GTG, or CAA sequence. In some embodiments, the 3' end of the target sequence is adjacent to the NGA, NAA, NGCG, NGN, NNGRRT, NNNRRT, NGCG, NGCN, NGTN, NGTN, NGTN, or 5'(TTTV) sequence.

[0637] It should be understood that the numbering of specific positions or residues in a particular sequence depends on the particular protein and numbering scheme used. Numbering may differ, for example, between a precursor of a mature protein and the mature protein itself, and sequence differences between different species may affect numbering. A skilled artisan will be able to identify the individual residues in any homologous protein and in the respective encoding nucleic acid by methods well known in the art, for example, by sequence alignment and determination of homologous residues.

[0638] It will be apparent to those skilled in the art that in order to target any of the fusion proteins described herein to a target site, such as a site comprising a mutation to be edited, the fusion protein will typically be expressed with a guide RNA. As explained in further detail herein, the guide RNA typically comprises a tracrRNA framework that allows Cas9 to bind; and a guide sequence that confers sequence specificity to the Cas9: nucleic acid editing enzyme / domain fusion protein. In addition, the guide RNA and tracrRNA may be provided separately as two nucleic acid molecules. In several embodiments, the guide RNA comprises a structure in which the guide sequence comprises a sequence complementary to the target sequence. The guide sequence is typically 20 nucleotides in length. Based on the disclosure herein, it will be apparent to those skilled in the art that suitable guide RNA sequences for targeting Cas9: nucleic acid editing enzyme / domain fusion proteins to specific genome target sites. Such suitable guide RNA sequences typically comprise guide sequences that are complementary to nucleic acid sequences within 50 nucleotides upstream or downstream of the target nucleotide to be edited. Provided in the present disclosure are several examples of convenient guide RNA sequences suitable for targeting any of the provided fusion proteins to a specific target sequence.

[0639] Methods for using fusion proteins comprising a Cas9 domain and a cytidine deaminase or adenosine deaminase

[0640] Some aspects of the present disclosure provide methods for using fusion proteins or complexes provided in the present disclosure. For example, some aspects of the present disclosure provide methods, including contacting a DNA molecule encoding a protein of interest with any of the fusion proteins provided in the present disclosure, and contacting with at least one guide RNA, wherein the guide RNA is about 15-100 nucleotides in length, and comprises a sequence of at least 10 adjacent nucleotides, which is complementary to the target sequence. In some embodiments, the 3' end of the target sequence is adjacent to the canonical PAM sequence (NGG). In some embodiments, the 3' end of the target sequence is not adjacent to the canonical PAM sequence (NGG). In some embodiments, the 3' end of the target sequence is adjacent to the AGC, GAG, TTT, GTG, or CAA sequence. In some embodiments, the 3' end of the target sequence is adjacent to the NGA, NAA, NGCG, NGN, NNGRRT, NNNRRT, NGCG, NGCN, NGTN, NGTN, NGTN, or 5' (TTTV) sequence.

[0641] Additional Domains

[0642] The base editors described herein may include any domain that helps assist nucleobase editing, modification or alteration of nucleobases of polynucleotides. In some embodiments, the base editor comprises a polynucleotide programmable nucleotide binding domain (e.g., Cas9), a nucleobase editing domain (e.g., a deaminase domain), and one or more additional domains. In some cases, the additional domains may assist the enzymatic or catalytic function of the base editor, the binding function of the base editor, or as an inhibitor of a cellular mechanism (e.g., an enzyme) that may interfere with the desired base editing result. In some embodiments, the base editor may include a nuclease, a nickase, a recombinase, a deaminase, a methyltransferase, a methylase, an acetylase, an acetyltransferase, a transcription activator, or a transcription repressor subdomain.

[0643] In some embodiments, a base editor may comprise a uracil glycosylase inhibitor (UGI) domain. The UGI domain can improve the efficiency of a base editor comprising a cytidine deaminase domain, for example, by inhibiting the conversion of U formed by C deamination back to a C nucleobase. In some cases, a cellular DNA repair response to the presence of U:G heterodiploid DNA may result in a reduction in the efficiency of nucleobase editing in the cell. In such cases, uracil DNA glycosylase (UDG) can catalyze the removal of U from DNA in the cell, which can trigger base excision repair (BER), mostly resulting in the reversal of U:G pairs to C:G pairs. In such cases, BER can be inhibited in a base editor comprising one or more domains that bind to a single strand, block the edited base, inhibit UGI, inhibit BER, protect the edited base, and / or promote the repair of non-edited strands. Thus, the disclosure herein contemplates base editor fusion proteins comprising a UGI domain.

[0644] In some embodiments, the base editor comprises all or part of a double-strand break (DSB) binding protein as a domain. For example, the DSB binding protein may include the Gam protein of bacteriophage μ, which can bind to the end of the DSB and protect the DSB from degradation. See Komor, AC, et al., "Improved base excision repair inhibition and phage μ Gam protein with higher efficiency and product purity to obtain C:G-to-T:A base editor", Science Advances 3:eaao4774 (2017); the entire disclosure of this specification is incorporated by reference.

[0645] In some embodiments, the base editor comprises all or part of a nucleic acid polymerase (NAP) as a domain. For example, the base editor may comprise all or part of a prokaryotic NAP. In some embodiments, the NAP or part thereof incorporated into the base editor is a DNA polymerase. In some embodiments, the NAP or part thereof incorporated into the base editor has translesion polymerase activity. In some embodiments, the NAP or part thereof incorporated into the base editor is a translesion DNA polymerase. In some embodiments, the NAP or part thereof incorporated into the base editor is a Rev7, Rev1 complex, polymerase ι, polymerase κ, or polymerase η. In some embodiments, the NAP or part thereof incorporated into the base editor is a component of a eukaryotic polymerase α, β, γ, δ, ε, γ, η, ι, κ, λ, μ, or ν. In some embodiments, the NAP or portion thereof incorporated into the base editor comprises an amino acid sequence that is at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 99.5% identical to a nucleic acid polymerase (e.g., a translesion DNA polymerase).

[0646] Base editor system

[0647] The base editor system provided in the present disclosure comprises the following steps: (a) contacting a target nucleotide sequence of a subject polynucleotide (e.g., double-stranded DNA or RNA, single-stranded DNA or RNA) with a base editor system comprising a nucleoside base editor (e.g., an adenosine base editor or a cytidine base editor) and a guide polynucleotide (e.g., a gRNA), wherein the target nucleotide sequence comprises a targeted nucleobase pair; (b) inducing strand separation in the target region; (c) converting a first nucleobase of a target nucleobase pair in a single strand of the target region to a second nucleobase; and (d) cleaving no more than one strand in the target region, wherein a third nucleobase complementary to the first nucleobase is replaced by a fourth nucleobase complementary to the second nucleobase. It should be understood that in some embodiments, step (b) is deleted. In some embodiments, the targeted nucleobase pairs are a plurality of nucleobase pairs in one or more genes. In some embodiments, the base editor system provided in the present disclosure is capable of multiplexing multiple nucleobase pairs in one or more genes. In some embodiments, the multiple nucleobase pairs are in the same gene. In some embodiments, the multiple nucleobase pairs are in one or more genes, at least one of which is located at a different locus.

[0648] In some embodiments, the cleaved strand (nicked strand) hybridizes to a guide nucleic acid. In some embodiments, the cleaved strand is opposite to the strand comprising the first nucleobase. In some embodiments, the base editor comprises Cas9. In some embodiments, the first base is adenine, and the second base is not G, C, A, or T. In some embodiments, the second base is inosine.

[0649] The base editor system provided in the present disclosure provides a novel approach to genome editing, which uses a fusion protein containing a catalytically defective Streptococcus pyogenes Cas9, a cytidine deaminase, and a base excision repair inhibitor to induce programmable single nucleotide (C→T or A→G) changes in DNA without generating double-stranded DNA breaks, requiring no donor DNA template, and without inducing excessive random insertions or deletions.

[0650] Provided herein are systems, compositions, and methods for editing nucleobases using a base editor system. In some embodiments, the base editor system comprises (1) a base editor (BE) comprising a polynucleotide programmable nucleotide binding domain-and a nucleobase editing domain (e.g., a deaminase domain) for editing nucleobases; and (2) a guide polynucleotide (e.g., a guide RNA) together with the polynucleotide programmable nucleotide binding domain. In some embodiments, the base editor system comprises a cytidine base editor (CBE). In some embodiments, the base editor system comprises an adenosine base editor (ABE). In some embodiments, the polynucleotide programmable nucleotide binding domain is a polynucleotide programmable DNA binding domain. In some embodiments, the polynucleotide programmable nucleotide binding domain is a polynucleotide programmable RNA binding domain. In some embodiments, the nucleobase editing domain is a deaminase domain. In some cases, the deaminase domain can be a cytosine deaminase or a cytidine deaminase. In some embodiments, the terms "cytosine deaminase" and "cytid...

Claims

1. A method for editing a SERPINA1 polynucleotide comprising a single nucleotide polymorphism associated with alpha-1 antitrypsin deficiency, the method comprising contacting the SERPINA1 polynucleotide with a base editor complexed with one or more guide polynucleotides, wherein: The base editor comprises a polynucleotide programmable DNA binding domain and a cytidine deaminase domain, and wherein the one or more guide polynucleotides target the base editor to effectuate a change in a single nucleotide polymorphism associated with alpha-1 antitrypsin deficiency.

2. A cell produced by introducing into a cell or a progenitor cell thereof: A base editor or a polynucleotide encoding the base editor, wherein: The base editor comprises a polynucleotide programmable DNA binding domain and a cytidine deaminase domain; and One or more guide polynucleotides target the base editor to deaminase cytidine at nucleic acid position 1455 of the SERPINA1 polynucleotide.

3. A method of treating alpha-1 antitrypsin deficiency in a subject, comprising administering to the subject the cell of claim 2.

4. An isolated cell or cell population proliferated or expanded from the cell of claim 2.

5. A method of treating alpha-1 antitrypsin deficiency in a subject in need thereof, comprising administering to the subject: A base editor or a polynucleotide encoding the base editor, wherein: The base editor comprises a polynucleotide programmable DNA binding domain and a cytidine deaminase domain; and One or more guide polynucleotides target the base editor to effect a change in the cytidine at nucleic acid position 1455 of the SERPINA1 polynucleotide.

6. A method for producing hepatocytes or progenitor cells thereof, comprising: (a) introducing into a hepatocyte progenitor cell containing a single nucleotide polymorphism associated with alpha-1 antitrypsin deficiency, A base editor or a polynucleotide encoding the base editor, wherein: The base editor comprises a polynucleotide programmable nucleotide binding domain and a cytidine deaminase domain; and one or more guide polynucleotides, wherein the one or more guide polynucleotides target the base editor to deaminase the cytidine at nucleic acid position 1455 of the SERPINA1 polynucleotide; and (b) differentiating the hepatocyte progenitor cells into hepatocytes.

7. A guide RNA comprising a nucleic acid sequence selected from the group consisting of: 5'-CAAUCAUUAAGAAGACAAAAGGGUUU-3'; 5'-UCAAUCAUUAAGAAGACAAAAGGGUUU-3'; 5'-UUCAAUCAUUAAGAAGACAAAAGGGUUU-3'; 5'-GUUCAAUCAUUAAGAAGACAAAAGGGUUU-3'; 5'-UGUUCAAUCAUUAAGAAGACAAAAGGGUUU-3'; 5'-UUGUUCAAUCAUUAAGAAGACAAAAGGGUU-3'; 5'-UUCAAUCAUUAAGAAGACAAAG-3'; 5'-UUCAAUCAUUAAGAAGACAAAAGG-3'; 5'-UCAAUCAUUAAGAAGACAAAGGG-3'; or 5'-AAUCAUUAAGAAGACAAAGGGU-3'.

8. A guide RNA comprising 18, 19, 20, 21 or 22 nucleotides of the guide RNA of claim 7.

9. A protein-nucleic acid complex comprising a base editor and the guide RNA as described in claim 7.

10. A method of treating a genetic disorder in an individual in need thereof, comprising: administering a base editor or a polynucleotide encoding the base editor to the individual, wherein: The base editor comprises a polynucleotide programmable nucleotide binding domain and a deaminase domain; administering a guide polynucleotide to the individual, wherein the guide polynucleotide targets the base editor to a target nucleotide sequence in the individual; and When the base editor is targeted to the target nucleotide sequence, the nucleobase of the target nucleotide sequence is edited by deaminating the nucleobase, thereby treating the genetic disorder by changing the nucleobase to another nucleobase; wherein the nucleobase is in a protein coding region of the polynucleotide; and wherein the nucleobase is not causative of the genetic disorder.

Images